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615 Cards in this Set

  • Front
  • Back
3 components of nucleic acids
base (nitrogen), sugar (pentose), phosphate
Rings in a Purine
Rings in a Pyrimidine
Purine Nucleotides
Adenine and Guanine
Pyrimidine Nucleotides
Cytosine, Uracil and Thymine
What is the central dogma of molecular biology
DNA --> RNA --> Protein
What is the net charge on an ATP molecule
How do nucleotides bond to each other
Bonds in A-T (or A-U)
Bonds in C-G
What carbon is the glycositic linkage at in the ribose backbone?
What carbon is the site of phosphate addition in the ribose backbone?
What carbons establish polarity in the ribose backbone?
3' and 5'
What carbon is the phosphodiester bond at in the ribose backbone?
3' and 5'
What hyrdoxide group is different between ribose and deoxyribose
What is the difference between a nucleoside, nucleotide, and nucleic acid
Nucleoside=base+sugar, nucleotide=base+sugar+phosphate, nucleic acid=polymer of nucleotides
In what direction is the nucleotide sequence read?
5' -> 3'
What are the grooves in DNA?
Major and minor
What was an experiment that showed that DNA was the genetic material?
labeled proteins and DNA in a phage with radioactive labels, DNA correlated with virulence
What were the Griffith, Avery MacCleod and MacCarthy experiments?
Showed that genetic material from heat-killed bacteria could cause non-toxic bacteria to be toxic.
What is DNA supercoiling and what are the two forms it takes?
positive (overwound, unstable+left turns), negative (underwound, stable+right turns)
How can you untangle messy DNA
DNA treated with heat and base will do what?
Undergo strand separation
What do topoisomerases do?
Nick DNA
How do topoisomerases work as therapeutic agents
Ciproflaxin and novobiocin (antibiotics) inhibit bacterial gyrases, Etoposide and Doxorubicin affect eukaryotic type II topoisomerases in cancer treatment
What is the shape of the eukaryotic genome? Viruses? Bacteria
large condensed chromosomes, linear, and circular (supercoiled) for bacteria
How many chomosomes to humans have?
22 autosomes + XX or XY
What is chromatin?
Highly organized structure with histone proeins that bind and twist DNA around them to form nucleosomes
What is a histone?
a protein that forms the core of a nucleosome
What is a nucleosome?
A complex of 4 pairs of histones (H2A, H2B, H3, H4) referred to as the octamer, that DNA wraps around two times
What is a solenoid?
A 30nm chromatin fiber composed of 6 nucleosomes
What joins nucleosomes together
linker DNA of variable length and histone H1
How can histones be modified?
acetylation (activates), deacetylation (deactivates), methylation (activation and inactivation), phosphorylation (increases condensation), and ubiquitination
What is Cofin Lowry Syndrome
mutation in RSK (kinase that phosphorylates histones to further condensation of chromosome); mutation disrupts gene expression, leading to devel. Defects and mental retardation.
What are diseases that are involved in DNA methylation?
ICF syndrome, Rett syndrome
What is fragile-X chromosome and what causes it?
Most common heritable form of mental retardation-- trinucleotide expansion repeat in FRAX gene fragile site.
What is the centromere?
Non-coding region of attachment of p and q chormosome ends, site of spindle attachment during mitosis
What is the telomere?
non-coding region at each end of the chromosome, created by telomerase, prevents chromosome shortening in replication
What classes of sequences are there in the human genome?
Unique 1-10 copies per genome, middle repetetive (10-10,000) tRNA and rRNA genes, higly repetitive (>100K) non-coding regions
What is replication, transcription, translation?
DNA synthesis (DNA pol), RNA synthesis (RNA pol), protein synthesis (cellular ribosomes and tRNA)
What diseases are examples of a misregulation of DNA replication?
Cancer, viral disease
How can you experimentally differentiate between a DNA synthesis initiation protein and an elongation protein?
Use heat shock protein to stop synthesis; an initiation protein will stop replication after a lag time, an elongation protein will stop synthesis immediately
What direction is DNA sythesized?
How are nucleotides added to a growing DNA chain?
NTP covalently attaches to the 3' OH, and Ppi is released.
What do di-deoxynucleotides do?
Stop synthesis of DNA
The region of the genome serviced by one origin of replication
The complex of proteins that function at the replication fork
What is a theta structure?
A plasmid that has begun to replicate--it has a fork and it looks like an eye
What is primase?
an RNA polymerase, initiates replication by adding short complementary RNA primers to both strands of DNA
Okazaki fragment
Short segment of DNA synthesized between primers on the lagging strand
2 requirements for DNA replication
proteins, RNA synthesis
Four prokaryotic polymerases (not all)
Primase, DNA Pol I, DNA Pol II, DNA Pol III
DNA polymerase I
5' to 3' DNA synthesis, 3' to 5' proofreading, (okazaki fragments) 5' to 3' exonuclease replaces primers with DNA
DNA polymerase II
5' to 3' DNA synthesis, 3' to 5' proofreading
DNA polymerase III
5' to 3' DNA synthesis (major DNA polymerase), 3' to 5' proofreading
What are some features of the origin of replication?
9'mers and 13-mers bind DnaA, A-T rich to facilitate unwinding, protein binding sequences
What is DnaA
binds to 9-mers in eukaryotes to initiate synthesis
What do DnaB and DnaC do?
DnaB is a helicase, unwinds DNA after DnaC loads it on the DNA and lets go (ATP dependent)
What is DnaG?
Bacterial primase
What are SSBs?
Single-stranded binding proteins, protect ssDNA from degradation
What comprises the DNA Pol III holoenzyme?
Clamp loader, clamp (homodimer), core enzyme, DNA pol dimerizer
How is replication terminated?
tus proteins bind ter sites-- DNA Pol III and DnaB fall off, terminating replication
Why are topoisomerases important in replication
Because helicase would have to rotate the DNA 50 times/second to keep unravelling it
What are the eukaryotic DNA synthesis enzymes?
DNA pol alpha, DNA pol delta, DNA pol epsilon
DNA polymerase alpha
Primase, makes RNA primers+first few dNTPs, 5'-3' RNA and DNA synthesis
DNA polymerase delta
DNA elongation, 5'-3' DNA synthesis and 3'-5' proofreading
DNA polymerase epsilon
DNA elongation, exact function unknown
What is processivity?
The ability to synthesize a long stretch of DNA and not fall off
Process by which cell is induced to enter the S phase
pre-replication complex
Forms at origin of replication, necessary for licensing
Key proteins involved in licensing
ORC, Cdt, Cdc6/18, MCM proteins, Geminin
blocks licensing by (possibly) interacting with Cdt1, also disassembles replication machinery
Cdt1/Cdc6/18/MCMs complex
pre-replication complex, forms when Geminin is degraded and initiates replication
Eukaryotic helicase
Eukaryotic equivalent of DnaA
ORC, Dcc6/18, Cdt1
Eukaryotic clamp loader
Eukaryotic clamp
DNA pol dimerizer (eukaryotes)
Not known
Eukaryotic RNA removal
Ligase I
What is SV40
animal virus isolated from African Monkey, used as model for mammalian replication machinery
T antigen
key component of SV40 DNA replication machinery--acts as pre-replication complex
Single-stranded binding proteins, protect ssDNA from degradation
What does telomerase do?
prevents strand shortening because of lagging strand problems with synthesis
What end extends too far at the end of a chromosome?
End of DNA bound to itself by telomerase to cap chromosome
ss 3' loop invades the duplex and hybridizes
Telomerase activity
high in germ cells/embryonic cells; as differentiation increases, activity decreases
Abnormal telomerase activity
many cancer cells/immortalized cells reactivate telomerase
2 cell cycle proteins modified by viruses
p53 (temporarily prevents entry into S phase when DNA is damaged), Rb (binds and inhibits E2F, a TF that would otherwise induce expression of replication proteins)
Characteristics of SV40
causes tumors when injected into rodents, no sign of disease in humans, genome=ds circular DNA, model for eukaryotic replication
T antigen's effects in host cell
Binds p53, inactivating it; binds Rb, inactivating it; also acts as pre-replication complex
SV40 replication procedure
SV40 encodes LT, 2 LT complexes bind minimal origin, eukaryotic machinery engages replication (LT is helicase), topoisomerase separates+uncoils plasmids
Adenovirus activity (E1A and E1B)
E1A binds RB blocking its binding to E2F, E1B binds p53, inactivating it
Drugs that target replication
AZT, ddI and ddC, Acyclovir, Actinomycin, Acridine dyes, Daunorubicin
AZT mechanism of action
Azidothymidine, AKA Zidovudine, T analog lacking 3' OH, specific for HIV reverse transcriptase
ddI and ddC mechanism of action
Analogs that lack 3' OH--used to treat HIV
Acyclovir mechansim of action
G analog lacking 3' OH, specific for HSV and Herpes Varicella Zoster (chicken pox) DNA polymerase
Actinomycin, Acridine dyes, Daunorubicin mechanism of action
intercalating agents that incorporate into the DNA duplex and disrupt replication, transcription and recombination processes
Adenovirus genome
ds linear DNA, undergoes replication as circularized then linear DNA, contains 3' pTP
Rolling Circle replication
Herpes virus linear DNA ligates into a circle, replicates as a theta structure, then as a rolling circle.
What is a DNA polymorphism?
single or multi-base pair change in genetic code
What is the difference between a functional and a molecular polymorphism?
a functional polymorphism occurs within a gene and may affect its function; a molecular polymorphism (eg sickle cell anemia polymorphism) occurs close to a gene and may not affect its function-- it can often be used as a marker to identify the gene
What does a restriction enzyme do?
Recognizes and cleaves specific DNA "recognition" sequences
What is the difference between an endonuclease and an exonuclease?
endonucleases recogize dsDNA and cleave leaving blunt or a sticky ends; exonucleases recognize the ends of DNA strands and digest them--exonucleases are especially important in apoptosis
How do bacteria protect themselves from their own endonuclease activity?
By pairing restriction endonucleases with DNA modification enzymes (methylases)--methylated DNA will not be cut.
For the purposes of this course, what is a clone?
a segment of DNA of interest and its vector-- can be manipulated/isolated through use of restriction endonucleases
Is DNA negatively or positively charged? Why?
Negatively-- phosphate groups
What is the principle behind DNA gel electrophoresis?
using the charge on DNA (-) to move different sized fragments through a gel at different rates--large pieces migrate more slowly
What is the easiest way to denature and re-anneal DNA?
heating and cooling
What is Southern Blotting?
Assay for DNA by: restriction endonuclease digest, then gel electrophoresis, membrane transfer, and visualization with hybridized DNA probes
What is Northern Blotting?
Assay for RNA by: production and isolation of RNA, then gel electrophoresis, membrane transfer, and visualization with hybridized DNA (or RNA) probes
What is FISH?
Fluorescence in situ hybridization. Analysis of DNA (or RNA) expression/presence. Denature DNA in fixed tissue and probe using DNA probes
How is FISH useful to probe for translocation among chromosomes?
You can isolate chromosomes and probe them using labeled DNA probes and see if sections of chromosomes have translocated
What is the Philadelphia Chromosome?
A translocation between chromosomes 9 and 22 that leads to BCR-Ab1 fusion (resulting in chronic myologenous leukemia)
What is RFLP?
Restriction fragment length polymorphism-- differences in digested DNA fragment lengths. These can be electrophoresed for "DNA fingerprinting"
Why is Sir Alec Jeffreys famous?
He was interested in the idea of using repetetive DNA as a source of personal inforamtion (DNA fignerprinting). So there.
Why is Mary-Claire King famous?
she defined genes associated with inherited forms of breast cancer (BRCA1)
How does Sanger Dideoxy (DNA) sequencing work?
using dideoxy nucleotides (ddNTPs) which lack a 3' OH--therefore terminate replication, DNA is synthesized from ssDNA fragments in four reactions (each containing a particular ddNTP at low concentrations); synthesis will stop on select NTP at different lengths-- these can be separated on a gel/capillary and the relative concentrations of nucleotides quantified for sequencing
What is an oligo?
a chemically sythesized oligonucleotide
In what direction are oligos synthesized?
3' --> 5'
What is PCR?
Polymerase Chain Reaction-- DNA replication and amplification technique using DNA primers and Taq polymerase in a temperature-controlled reaction. Used to amplify DNA/analyze expression of DNA
What is a DNA microarray?
Technique using spotted probes and labeled DNA to measure expression levels of RNA in tissues, whatever
How might a microarray be useful in studying cancer?
Determining what genes are upregulated in cancerous tissue--could be used to characterize malignant vs. nonmalignant, test for prognosis of metastasis, analysis of chemo (or other) treatment, determining cancer genes, and more!
How might microarrays be useful in studying develomental anomalies?
difficult-to detect clinically, but may be due to microdeletions/microtranslations, analysis of small changes in DNA may allow more robust diagnostics
What is the goal of pharmacogenetics?
To understand and direct drug targets and drug efficacy based on genetic SNPs in families/individuals
How could pharmacogenetics be used to tailor therapies?
allows genotype-specific drug targeting; eg in cytochrome p450 gene polymorphisms --> disease but gene chip analysis can determine if patients will respond to particular drugs
What is recombination?
the exchange of genetic material between separate DNA duplexes, which may yield distinct products
What genetic processes involve recombination?
meiosis, antibody and T cell diversity, viral integration into genome, transposon integration
What are the two types of recombination?
homologous (genetic) and site-specific
What are the major differences between homologous and site-specific recombination?
DNA length: homologous preserves; site-specific doesn’t; location: homologous occurs between homologous sequences; timing: homologous generally meiosis and repair, site-specific in VDJ, host integration
Is genetic information lost in homologous recombination?
What are the overall steps in homologous recombination?
Initiation: ds break and creation of 2 free ends; pairing "invasion" of homologous DNA; DNA synthesis: polymerization and ligation; resolution of the intermediates
What is NHEJ?
Non-homologous end joining; repairs ds breaks
What does Rec BCD do in recombination?
helicase and endonuclease activity
What does RecA do in recombination?
ssDNA binding protein, mediates invasion during homology search
What is Rad51?
a critical eukaryotic homolog of RecA--loss or failure to assoc. w/ Brca1 or Brca2 can result in accumulation of DNA damage
What do Ruv A, B, and C do in recombination?
promote resolution of the intermediaries (eukaryotic homologues function in apoptosis and chromatin remodeling)
What enzymes are important in homologous recombination?
Rec BCD- ds break and pairing; RecA- ssDNA binding, invasion; Ruv A,B,C- promote resolution
What enzyme is critical for site-specific recombination?
What are the two types of transposition?
transposon (donor DNA-->DNA intermediate-->DNA reintegration) and retrotransposon (Donor DNA-->RNA-->DNA intermediate-->reintegration)
What are the major constituents of a transposon?
flanking (host) DNA, mobile element (w/ regulation/targeting sequences)
What is the difference between replicative and non-replicative (conservative) transposition?
Replicative (involves complex transposons) generates a copy of the transposon and results in 2 copies of the segment through site-specific recombination; non-replicative excises the DNA segment for the transposon and inserts it into a new site in the genome
What is an insertion element
simplest transposon unit--infect prokaryotes and eukaryotes, encode transposase and have inverted terminal repeats, flanked by direct repeats; undergo random integration into genome
What is the difference between an ITR and a DR (in a transposon)?
ITRs are ~50 bps characteristic of IS that mark the boundaries of the IS, DRs are generated because of jagged cleavage of target DNA and insertion, then repair of gaps (they are length, but not sequence-specific)
What are the two functions of transposase in IS translocation?
Makes a ds blunt cut between the ITR and DR of transposon, makes a ds jagged cut in target DNA
What are complex transposons?
additional genes flanked by two ISs, generally infect bacteria, eg provide antibiotic resistance
What are the major differences between insertion sequences and complex transposons?
IS uses non-replicative (conservative) transposition; complex us replicative, IS is moved, complex is replicated through ds nick, ss ligation, replication, and recombination
What is a retrotransposon?
like a complex transposon, but with ability to reverse transcribe its own mRNA to DNA (encode reverse transcriptase) mainly infect eukaryotes; major source of change in eukaryotic genome
What are the two types of eukaryotic nonviral retrotransposons?
What are the differences between LINEs and SINEs
L=Long interspersed elements, S=short; LINEs are transcribed (by RNA PolII)--> have ORFs and generate proteins, SINEs don't, have no ORFs (are transcribed by RNA PolIII)
What are some uses of transposons?
gene disruption, change activity at a promoter, deletions/inversions, insertion of a harmful or advantageous gene
What are Alus and what is L1?
Alus are SINEs (eg NF1) and L1 is a LINE
What are examples of mutagens?
Ionizing or UV radiation, chemical mutagens, pathogens, transposons
What is the difference between ionizing and UV radiation mutations?
UV radiation-->pyrimidine dimers-->ds break vs. x-ray-->???-->ss break
What are the two types of tautomeric shifts in base pairs leading to mutation?
A-->Aimino binds C instead of T; T-->Tenol binds G instead of A
How does 5-Bromouracil lead to bp mutation?
5-BUketo binds to A, but quickly changes to 5-BUenol,, which binds to G
How does bp mutation relate to cacer?
mutations can lead to cancer, but can also be important to block DNA replication/cancer
How does the formation of pyrimidine dimers disrupt DNA?
intra-strant covalent link between thymines (cytosine?) (C6-C or C4-C) distorts the duplex, corrected by excision
How does nitrous acid cause mutations in DNA?
deaminates bases (Adenine-->Hypoxanthine and binds with Cytosine; cytosine-->uracil and binds with adenine)
How can methylation cause mutations in DNA?
methyl group adds onto base (onto O in guanine) and distorts double helix; makes site sensitive to hydrolysis and can cause loss of a base
How does oxidation cause mutations in DNA?
thymine-->thymine glycol (disrupts structure/H-bonding)
What is an example of a bulky adduct that mutates DNA?
cis-platin crosslinks DNA duplexes; adds a platinum product that has two binding sites
What proteins in prokaryotes are involved in DNA repair?
Lex A, Rec A (SOS response)
What proteins are involved in eukaryotic DNA repair?
ATM or p53
How is prokaryotic DNA repair regulated?
Lex A inhibits DNA repair by binding to inhibitory sites; Rec A recognizes DNA damage and cleaves LexA and signals need for repair, Lex A falls off and DNA repair proteins are made
How is eukaryotic DNA repair regulated?
p53 and ATM proteins recognize DNA damage and signal for help, p53 halts the cell cycle and activates DNA repair genes; DNA damage-->ATM-->p53-->DNA repair proteins are activated
What is the difference between DNA damage and mutation?
they are not the same-- DNA can be damaged in a number of ways, but it is in the repair of DNA that the sequence can be permanently altered (many repair mechanisms are imprecise or error-prone)
What are the different repair systems and the enzymes involved?
DNA pol proofreading, DS break repair --NHEJ, mismatch repair, chemical modifications--O6 MGMT, BER; Repair of UV Radiation Damage--NER, Photolyase, Translesion; Post-replication recombination repair
How does DNA Pol I proofreading work?
As DNA Pol synthesizes DNA, proofreads, using 3' --> 5' exonuclease activity; if it recognizes a mismatch, it cleaves it out and replaces it w/ proper dNTP
How does DNA Pol cut out mismatches
Transfers growing strand from polymerization site to exonuclease site, cuts it out, goes back to synthesis
What is NHEJ?
Non-homologous end joining; joining non-homologous ends to repair double-stranded breaks
What proteins are involved in NHEJ?
Ku and DNA-dependent kinase bind the free ends of dsDNA and form a synapse-- DNA PKc recruits Rad50/MRE11/NBS1, which chews off the damaged ends
What is the end result of NHEJ?
loss of some base pairs (chewed off until shrot homology regions are found) but religation of DNA
What enzymes are involved in prokaryotic mismatch repair?
Mut S, Mut L, Mut H, exonucleases, DNA Pol III, ligase
What is the sequence of events in prokaryotic mismatch repair?
Mut S dimer binds to the mismatch, Mut L dimer binds to Mut S, sending Mut S translocating to a GATC site, Mut H joins the complex at GATC, cleaving the unmethylated strand. Exonucleases degrade the strand and then DNA Pol III fills in, ligase finishes the job.
What are the eukaryotic enzyme equivalents to prokaryotic mismatch repair?
Mut S = MSH proteins, Mut L = MLH proteins, Exonuclease = exonuclease, DNA Pol III = DNA Pol delta, Ligase = ligase, SSB's = RPA.
What are the major differences between prokaryotic and eukaryotic mismatch repair?
The strand discrimination process is unknown--GATC site isn't used (no Mut H equivalent known)
What are some important mismatch repair defects to know about?
MSH2 defects can have harmful consequences: DNA Pol slippage can create oncogenes, Cis-platin is used to block cancer; MSH2 problems can block this effectiveness.
How is improper methyl modification repaired?
O6 Methyl-Guanine-Methyl transferase transfers the improper methyl group onto a cysteine group on itself
What is Base Excision Repair and how does it work?
A chemically damaged/incorrect base is replaced with the proper one; glycosidase cleaves the base, then AP endonuclease cleaves off the sugar, then DNA Pol fills in the gap, then ligase ligates.
What DNA polymerases are involved in BER?
DNA Pol I (prokaryotes), DNA Pol beta (eukaryotes short patch) DNA Pol delta (eukaryotes long patch)
What is the difference between short patch and long patch BER?
short patch--repair of one improper NT, long patch--repair of multiple improper NTs
What is the sequence of events in eukaryotic DNA repair?
Genotoxic agents/Spontaneous damage/Replication errors-->DNA damage-->cell cycle arrest and repair by BER, recombination, mismatch repair-->mutation (if not repaired) or restored DNA (if repaired) or Apoptosis (if irreparable)
Lecture 12
Finishing Repair
How can DNA damage become a mutation?
if repair mechanisms don't work and the damaged cell does not undergo apoptosis, and the residual damage is carried by a germ-line cell, the damage can become a mutation
What are the main approaches possible to correct DNA damage?
cell cycle arrest, then BER, NER, recombination, mismatch repair, direct reversal, apoptosis as a last resort
What is NER?
nucleotide excision repair; process by which large DNA structural damage is repaired
What proteins are involved in prokaryotic NER?
Uvr proteins
What are the two main types of eukaryotic NER?
global genome repair and transcriptional coupled repair
How much damage can be accomplished by eukaryotic NER?
up to 9 kb of DNA
What is Xeroderma Pigmentosa?
deletion of XP genes-- 8 genes can be affected, causing an inability to repair DNA damage due to UV exposure (more common to get skin cancer)
How does prokaryotic NER work?
Uvr A protein, bound to DNA and scanning, detects deformation of DNA; Uvr A recognizes damage and binds there, Uvr B recruited and binds Uvr A, Uvr A is released in ATP-dependent fashion, Uvr C binds Uvr B at site of damage, Uvr C nicks 3-4 bp on one side, 4 bp on other side, Uvr B and C are released, Uvr D binds damaged area and helicase activity unwinds, releasing damaged strand; DNA Pol I and ligase repair gap
What does Uvr C do?
recognizes Uvr B on damaged DNA and nicks 3-4 bp and 4 bp on either side, then dissociates
What does Uvr D do?
helicase--recognizes damaged and (Uvr C) nicked DNA and unwinds it, freeing damaged strand as oligos that can be degraded
How does eukaryotic global genome repair work?
initial damage recognition, proteins bind and cleave DNA, oligo is removed and DNA re-synthesized to fill gap
What proteins are necessary in eukaryotic global genome repair?
XPC and hHR23B dimer binds to damaged and distorted DNA, TFIIH and other proteins bind to the complex and join site of damage, XPB and XPD are subunits of TFIIH and helicases, RPA and XPA bind and DNA is furtherunwound; XPG and XPF/ERCC1 are nucleases recruited to cleave DNA, DNA pol delta/epsilon fill in gap and ligase seals it
What do XPB and XPD do?
subunits of TFIIH, they are helicases that function in global genome repair
What do XPG and XPF/ERCC1 do?
they are nucleases that celave DNA and remove damaged nucleotides
What DNA pols are important in global genome repair?
DNA pol delta and epsilon
What is transcription coupled repair (TCR)?
a version of eukaryotic nucleotide excision repair; RNA pol can stall when it finds damage in the genome, CSA and CSB displace RNA Pol and recruit TFIIH to damaged DNA
What proteins are different between global genome repair and TCR?
RNA Pol recognizes damage and CSA and CSB bind to replace it, then recruit TFIIH (XPC and hHR23B are damage-recognizing proteins otherwise); from here XPB and XPD are the same (helicases) and XPA and RPA continue to lead down same path.
How is UV damage repaired?
translesion--DNA polymerases repair the damage
What proteins repair UV damage?
DNA polymerases (alternative to normal DNA pols)-- prokayrotes use DNA Pol V (Umu C and Umu D) and DNA Pol IV (Din B), eukaryotes use DNA Pol eta, DNA Pol zeta, Din B homolog
Why is UV damage repair error-prone?
a different subset of DNA pols are used to repair DNA, these have higher rates of misincorporation (but this is still better than having a gap in the DNA)
How does DNA Pol V translesion repair work?
DNA Pol III comes upon a lesion and falls off (cannot read through damage), Umu C and D are recruited to site and form dimer (=DNA Pol V), DNA Pol V is able to polymerize past lesion and replication continues (but Pol often inserts the wrong bases)
How does DNA Pol IV work?
Similar to DNA Pol V translesion repair, but leaves out nucleotides during polymerization, often leading to deletions which may result in frameshifts
What are the three main options for eukaryotic translesion synthesis?
DNA Pol eta, DNA pol zeta, Din B1
What is the process of eukaryotic translesion repair?
DNA Pol stalls at position of DNA damage and falls off, repair DNA pols are recruited
What are the characteristics of DNA Pol eta repair?
usually inserts proper nucleotides
What are the characteristics of DNA pol zeta repair?
requires Rev 1 and is error-prone
What are the characteristics of Din B repair?
insterts (often wrong) nucleotides
What causes xeroderma pigmentosa?
defects in XP genes, inability to repair damage caused by UV rays, higher susceptibility to skin cancer
What causes XP variant (XPV)?
defect in DNA pol eta (RAD30) which incorporates As opposite T-T dimers-- uses error-prone translesion repair. Has same symptoms as XP but excision repair is normal
What is Cockayne Syndrome?
defect in CSA and CSB (of TCR), leads to excessive cell death, neurological degeneration and stunted growth
What is post-replication recombination repair?
repairs ss gaps on a DNA strand whose complementary strand has large adducts, uses second genomic copy of damaged strand to synthesize a complementary one
What are the steps involved in post-replication recombination repair?
DNA Pol III stops at the lesion, Rec A facilitates invasion of the damaged strand into duplex of second chromosomal copy, DNA Pol I fills in the gap with complementary nucleotides
How does post-replication recombination repair work?
a gap is left in a newly synthesized strand where damage has taken place; in a poorly-understood process DNA Pol somehow copies nucleotides from the parental strand to fill in the daughter's gap
define a gene
the DNA containing information needed for some product (may be protein or RNA), also includes control sequences
What is the main enzyme involved in RNA synthesis?
multisubunit enzyme RNA Pol
How is the site of the initiation of transcription numbered?
+1 (downstream is +n, upstream is -n)
What are the two main types of RNA?
coding (mRNA in AA code) and non-coding (never become proteins)
What types of non-coding RNA are there?
rRNAs, snoRNAs (parts of ribosomal complex), tRNAs (translation), snRNAs (splicing), others: miRNAs, siRNAs (diverse functions in X-inactivation, gene expression and chromatin structure)
Does RNA act as a single strand?
No-- there are secondary and tertiary structures of RNA
What are some secondary structures of RNA?
stem-loop (large loop loop with double helical stem region with base pairing), hairpin (larger regions of base pairing with smaller loop)
What are some tertiary structures found in RNA?
pseudoknot, others
What do tertiary structures give to RNA?
specify function, influence stability, regulation (turnover), define phylogeny
Give me an overview of transcription
rNTPs are polymerized in a 5'->3' direction (phosphodiester bond formation catalyzed by RNA Pol), phosphate released in process (rxn direction driven by PPi cleavage)
What is the non-template strand?
the coding strand, since the template is complementary to both the RNA and the non-template
What are the three key steps in RNA transcription initiation?
polymerase binds to promoter sequence, forming closed complex, RNA Pol melts DNA duplex near transcription site, RNA Pol catalyzes formation of phosphodiester bond between two rNTPs
What is the closed complex?
RNA Pol bound to DNA duplex at transcription initiation site-- Pol is not yet working
What is the open complex?
RNA Pol has melted DNA duplex, forming transcription bubble
What is the transcription bubble?
ssDNA made as RNA Pol moves along DNA synthesizing RNA
How does RNA elongation work?
RNA Pol moves down template strand in 3'->5' direction, melting DNA and adding rNTPs to growing RNA chaing-- transient DNA-RNA hybrid exists at region of polymerization
How is transcription terminated?
once a stop site is reached, RNA Pol releases completed RNA and dissociates from DNA duplex
How are prokaryotic genes organized?
operons-- functional clusters of genes, engage in simultaneous transcription and translation
How are eukaryotic genes organized?
generally no clustering (some exceptions e.g. beta-globulin), primary transcript is processed (5' and 3' modifications and splicing) and RNA is transported to different regions of nucleus and cytoplasm
What are the main subunits of prokaryotic RNA Pol?
beta, beta-prime, alpha, omega, (delta-->specificity)
Lecture 13
Eukaryotic Transcription
What does the prokaryotic RNA pol haloenzyme consist of?
beta, beta prime, 2 alphas, omega (delta)
What is the core enzyme?
the beta and alpha subunits
What is the specificity/initiation subunit?
What subunit is necessary for loading?
Is eukaryotic RNA pol more or less complicated?
Do yeast and bacterial RNA pols have similar stuructures?
yes-- but yeast has 10 additional subunits that are not part of bacterial pol
Are eukaryotic genes organized into operons?
What is an operon?
a group of genes contolled by one common promoter (operator)
What in eukaryotic transcription replaces the function of the delta subunit in prokaryotic transcription?
multiple transcription factors (TFs) and TBP (TATA binding protein)
What are some examples of promoters?
GC-rich region, box A, box B, box C, TATA box
How many eukaryotic RNA polerases are there?
three-- Pol I, Pol II, Pol III
What does RNA Pol I do?
nuclelolar, pre-RNA (28S, 5.8S, 18S)
What does RNA Pol II do?
trnascribes protein-coding genes (mRNAs) and also a few non-coding RNAs (especially alpa-amanatin sensitive)
What does RNA Pol III do?
non-nucleolar tRNAs, 5s rRNA and other small RNAs
What is alpha-amanatin?
a bi-cyclic, octapeptide that potently inhibits RNA Pol II; comes from the death cap mushroom
What is the Pol II CTD?
C-terminal domain of Beta prime subunit that consists of a series of 7 repeated AAs that can be modified by posphorylation
When is POl II CTD phosphorylated?
it begins transcription phosphorylated but becomes highly phosphorylated as transcription proceeds. If the repeats are deleted the Pol doesn't work at all
What does RNA Pol I transcribe?
one RNA transcript from the rRNA gene-- 45S
What is the 45S RNA product?
pre-ribosomal RNA. It is cleaved into 18S 5.8S and 28S rRNA
What are the main transcription components of the 45S RNA gene?
promoter= GC rich, upstream element=UCE, transcription factors=UBF and SL1 (multimeric factors including TBP and TFs)
What is the RNA Pol I promoter?
GC-rich region
What is the upstream control element for RNA Pol I?
UCE-- important for transcriptional efficiency, but not essential for transcription
What is the core promoter region for RNA Pol I?
necessary for transciption, spans the region that includes the start of initiation
What is the order of assembly at the RNA Pol I promoter?
transcription factors determine the start site of RNA Pol I: UBF1 binds the GC-rich sequence of the promoter (UBF1 contains histones H3 and H4), SL1 binds cooperatively with UBF1, by an unknown mechanism there is a looping of UCE toward the promoter, Pol I associates and begins transcription
How is the 45S precursor processed?
step 1: 5' is cleaved off, step 2: 18S is cleaved off, step 3: 18 S is trimmed, step 4: 5.8S is cleaved off, step 5: 5.8S is attached to 28S
Does the 45S precursor go through mRNA splicing?
No--its cleavage is special somehow
What does RNA Pol III do?
transcribes 5S rRNA (for ribosomes) and tRNA
What are the components of RNA Pol III transcription?
5S: promoter=box A + box C (intragenic), TFs=TFIIIs; tRNA: promoter=box A and box B (intragenic), TFs=TFIIIs
What is an intragenic promoter?
a promoter that is within the product of a gene
Where are the promoters located for RNA Pol III?
downstream of the start point
What is the order of assembly at the RNA Pol I promoter for 5S transcription?
TFIIIA binds box A, TFIIIC binds TFIIIA and box C, TFIIIB binds TFIIIC and recruits RNA Pol III to the complex
What is the order of assembly at the RNA Pol I promoter for tRNA transcription?
TFIIIC binds box A and and box B, TFIIIB recruits RNA Pol III to the complex at the promoter
How is the 5S transcript processed?
it isn't--gets transported to nucleolus, then cytoplasm for ribosome assembly
How is tRNA processed?
endonuclease cleaves out an intron in the middle leaving two half molecules that are spliced together; extensively chemically modified, non-templated CCA is added to 3' end
What are snoRNAs?
small nucleolar RNAs-->factories that contribute to processing of pre-RNAs
Why does adenovirus hijack host RNA Pol III machinery?
it uses RNA Pol III for transcription of part of the viral genome, to generate VRNA, non-coding RNAs similar to tRNAs
How does adenovirus hijack RNA pol III?
TFIIIC1 binds the A box, TFIIIC2 binds the B box, TFIIIB recruits RNA Pol III to initiate transcription
What does RNA Pol II do?
transcribes mRNA, the only RNA that gets translated into protein
Why are multiple regulatory mechanisms necessary for RNA Pol II?
it has to recognize and transcribe from thousands of different promoters for tons of genes
What are some common features of RNA Pol II promoters?
TATA box (hogness box), CpG islands and Initiator sequence (I), Downstream Promoter Elements (DPE), Upstream Elements, Enhancers
Lecture 14
Eukaryotic Transcriptional Initiation
What are four main sequence classes that recruit Pol I?
TATA boxes, Initiators, CpG islands, DPEs
What is a TATA box?
a transcriptional promoter containining ten highly conserved nucleotides in positions -35 to -25
What does "consensus sequence" mean?
in a TATA box, it's the conserved sequence of 8 bp that is very similar, beginning with TATA
What are non-TATA initiation sequences?
Initiators (alternative promoter elements) have specific start sites, CpG islands, DPEs (downstream promoter elements)
How do CpG islands work?
they have less precise start sites than TATA boxes-- begin transcription of Pol II genes close by, have multiple 5' ends, often occur at housekeeping genes
What are the main categories of Pol II transcription factors?
BTFs (basal transcription factors) AKA General Transcription Factors, and STFs (stimulatory transcription factors)
What do BTFs do?
bind near promoter region to initiate transcription, specifically act to get Pol II started
What are some examples of BTFs?
How do TBPs work?
recognize sequence and bind in minor groove of DNA, cause DNA to bend, recruit and position Pol II; TAFs are TBP-associated factors
How do STFs work?
they bind the enhancer, affecting the initiation of transcription, also affect the frequency with which a particular gene is transcribed; regulate the activation of Pol II by binding upstream from the promoter
What is the usual structure for a TF?
multiple distinct polypeptides
How do TFIIDs work?
TFIID=TBP+TAFs; TBP recognizes and binds the promoter (often a TATA box) and recruits TAFs, forming TFIID complex (at least 11 other polypeptides make it up)
What is the cascade of TF interactions in TFIID signalling?
TFIIA displaces repressors, TFIIB binds TFIID, TFIIF recruits RNA Pol II to the pre-initiation complex, TFIIE is part of the complex, TFIIH is a helicase that unwinds the duplex; also a kinase that phosphorylates RNA Pol for elongation
What does RNA Pol II need to initiate transcription?
general transcription factors TFII, for step-wise assembly of ATP-dependent open-complex; net is 60-70 polypeptides, 3MDa mass
What is the general cascade for pre-initiation complex formation/transcription?
pre-initiation complex formation-->initiation-->transcription (CTD phos)-->dissociation of TBP/TAFs-->elongation
What is the step-by-step process of TBP initiation of transcription?
TBP binds to TATA box in minor groove, causing bending of DNA, dimeric TFIIB joins TBP at TATA box, pre-formed complex of Pol II (un-phosphorylated CTD) and TFIIF associates just downstream of TP-TFIIB at TATA box, TFIIE joins Pol II, TFIIH (helicase/kinase) binds to form pre-initiation complex, ATP is hydrolyzed to phosphorylate CTD, which allows elongation, polymerization occurs, release of general factors (except TBP)
What does phosphorylation of the CTD of RNA Pol II do?
stabilizes it for transcription, allowing it to keep moving down DNA
What is the minimal initiation complex?
TBP, TFIIB, PolII-TFIIF-- it functions to allow Pol II to get the first and second nucleotides going
What is the active initiation transcription complex?
the TBP+TFIIB+PolII-TFIIF+TFIIH+TFIIE (TFIIH is needed to really get things going); ATP is hydrolyzed and elongation begins
What is the elongation complex?
after all TFs have bound, ATP is hydrolyzed and TBP and TFIIB are left behind, TFIIE and TFIIH dissociate and just TFIIF-Pol II cruise down the track
What are enhancers?
regions separate from the promoter that bind STFs to enhance transcription at the promoter (beyond basal TFs)
What are the main types of STF interactions?
associate with BTFs to stabilize complex, directly associate with RNA Pol II, remove histones, physically bend DNA
What are co-activators (and co-repressors)?
STFs that interconnect TFs and do not bind DNA directly
What are the regulatory elements in the IFN-beta enhancer?
four control elements (HMG1) bind simultaneously, three heterodimeric regulators, highly cooperative with HMG1 (high mobility group 1) also bind; HMG1, architectural factor, binds major groove and bends DNA
How might upstream co-activators affect TFs?
they may contact TAFs on TFIID or TFIIB, and be involved right at start of initiation
How might upstream co-activators affect histones?
they may acetylate histone tails, leading to more open chromatin and aiding in transcription
How might a co-repressor affect transcription?
some co-repressors contain a histone deacetylase, other co-repressors reverse other catalytic activity, or structurally exclude RNA pol or TFs
What is an example of a co-repressor complex?
Ume6 (DNA binding), Sin3 (co-repressor), Rpd3 (deacetylase)
Lecture 15
Transcription Factor Domains
What are some possible modular domains of transcription factors?
protein binding domain (AKA activation domain), dimerization domain, DNA binding domain, bubble bath domain
What are some common motifs found in TF domains?
zinc finger, helix-loop-helix, homeodomain, leucine zipper
Can multiple domains exist on one TF?
sure. A TF may have, for example, a DNA binding domain, a conecting domain and an activating domain
How is the Gal4 paradigm useful to understand multiple functional domains of TFs?
Gal4p has DNA binding and activation domain, deletion mutants show that domains are discrete and can operate independent of each other
What do DNA-binding domains do?
recognize specific DNA sequences
What do Activation domains do?
interact with other proteins, they are often acidic
What do Flexible (linker) domains do?
located between the DNA-binding and activation domains, help facilitate spacing changes
How might chimeric, novel TFs be possible?
by combining diverse DNA-binding domains with activating domains
What are some possible modular domains of transcriptional repressors?
DNA-binding domains, repressor domains in binding proteisn
What would a mutation in a repressor cause?
abnormal activation (transcription) of a gene
How is WT1 a good example for a repressor (or activator of protein expression)
WT1 functions as a repressor at the EGR1 (early growth response 1) locus, repressor is mutated in pediatric kidney tumor. WT1 doesn't interfere with/occlude binding of activators at locus (also has SRF/TCF binding site, AP1 binding site), but balances amount of transcription at the EGR1 locus
What is highly conserved in DNA-binding proteins?
certain structural motifs (e.g. homeodomains, zinc-fingers, winged-helix (forkhead), helix-loop-helix, leucine-zippers (basic zippers))
What are the main forces that stabilize binding with DNA?
specific H-bonding, van der Vaals interactions, also sugar-phosphate and minor groove atoms can contribute
What is a common structural motif that recognizes the major groove of DNA?
helix-turn-helix: recognition helix binds to DNA, helix-turn-helix supports, stabilizes DNA-recognition helix interaction
What structural motif is seen in TFs often associated with developmentally important genes?
What residues coordinate binding in zinc-finger domains?
cysteine or histidine
How is the leucine zipper held together?
the basic regions of bZIP are held together by dimerization with the adjacent zipper region
What are the main structural components of a lecuine zipper?
basic regions bind DNA, leucine residues (repeated every 7 AAs) create hydrophobic face, this interaction contributes to protein stability and allows tight association between subunits
What are the main strutural components of HLH proteins?
HLH TFs have regions corresponding to helix 1 and helix 2 separated by a loop of 10-24 AAs. B-HLH have a basic region adjacent to helix 1
How can HLH proteins increase DNA affinity?
by dimerizing-- B-HLH proteins can dimerize and bind to DNA, non-basic HLH proteins can prevent DNA-binding (?)
How can heteroximerization increase the possibilities of TFs?
by allowing for many different TF activities to combine--3 factors can create 6 combinations; inhibitory factors can prevent binding (repressing activity); negative and positive regulation can occur…
What is CREB?
cAMP response element binding protein, stimulatory TF that activates genes induced by cAMP
What are the main players in CREB signalling?
cAMP, CRE, CREB, CBP, PKA, target
What does CRE do?
cAMP response element, upstream element (DNA sequence) bound by CREB
What does CBP do?
CREB-binding protein, co-activator that binds phosphorylated CREB and recruits basal complex to the promoter-- pt mutations in CBP can cause Rubenstein Taybi Syndrome (mental retardation, heart defects, hirsutism)
What is the cascade of signaling in CREB activation
cAMP levels rise, PKA is activated and phosphorylates CREB, phosphorylated CREB binds CRE, phosphorylated CREB binds CBP which recruits basal complex to bind TATA box/promoter, transcription is activated
What are some mechanisms to regulate the activity of TFs?
protein synthesis, (de-)phosphorylation, ligand binding, cleavage to release factor, release by inhibitor, change of partner
What does the SV40 enhancer look like?
2 side-by-side 72 bp elements-- containing multiple GC boxes that are recognized and bound by host TFs such as SP1, causing activation of early genes in SV40 genome
How does transcription start in SV40?
host machinery begins, then viral machinery takes over; transcription activation of SV40 early genes causes transcription of Large T and Small T antigens
How is gene transcription switched in SV40?
Large T antigen ultimately inhibis early gene transcription and activates late gene transcription
How does adenovirus transcription work in hosts?
host TFs recognize adenovirus sequences and activate transcription of immediate early genes including E1A. E1A activates transcription of early genes, including those involved in DNA replication. After replication, activation of late genes occurs
How does Herpes Simplex Virus transcription work?
HSV protein VP16 directly associates with host Oct1 to activate immediate early genes (including ICP4 and ICP0). IPC3 and IPC0 inhibit IE genes and activate early genes, which include TK and DNA pol; late genes are activated after DNA replication begins
What does VP16 do?
HSV transcription factor, packaged in the viral protein, that directly associates with host Oct1 to activate IE genes
How does HIV control transcription?
HIV genome has upstream enhancer, core enhancer and promoter, upstream and core enhancers and promoter all have binding sites for cellular TFs that can activate transcription of HIV genes
What is the difference in stop signals between transcription and translation?
translation stops at stop codons-- transcription doesn't have an equivalent
How does prokaryotic transcription stop?
several mechanisms, rho-depedent or independent
How is eukaryotic transcription stopped?
a different mechansim for each Pol
How does Pol I stop?
a termination factor binds to an 18 bp terminator sequence, Pol reaches termination factor and stops >1000 bp downstream of 3' end of mature transcript, 3' end of transcript is generated by post-transcriptional cleavage
How does Pol III stop?
Pol III terminates after a U-encoding series, embedded in a GC rich region (does not require termination factors), generally stops after the second U of series but sometimes after 3rd or 4th
What is a model for Pol III transcription termination?
inherent decreased thermal stability of AU base pairing in transcription bubble
Lecture 16
Where does RNA Pol II terminate?
imprecise-- can terminate at multiple sites over 0.5-2kb beyond poly-A signal. Termination is coupled to 3' cleavage near poly-A site
How does pausing fit into RNA Pol II function
Pol II can pause, allowing re-initiation of transcription without delay for assembly of initiation complexes; anti-termination can be important
How is pausing important in heat shock genes?
RNA Pol II normally pauses after 25 nucleotides, does not terminate-- upstream binding sites for heat shock transcription factor (HSTF) and HSTF binds during times of stress/denaturing conditions, kicks Pol II into gear
Does RNA Pol II have high or low processivity?
after first 50 nucleotides, incredibly high processivity-- important because
What is an HIV anti-termination factor?
How does HIV tat work?
Pol II transcribes genome, including tat, which binds to RNA TAR sequence-- tat is necessary for elongation by Pol II
What does the HIV TAR protein do?
TAR has stem-loop structure, binds Tat and Cyclin T; Cyclin T interacts with CdK9, phosphorylates CTD of Pol II and promotes elongation
Without tat, what would happen to RNA Pol II in HIV transcription, and why does this make it a good drug target?
without tat, transcription would stop after about 50 nucleotides-- if you can block tat function, you can block HIV transcription
How is HIV Tat transcribed?
It's downstream of TAR-- 1 in 10 of TAR transcripts also transcribe Tat
What are the main differences between RNA Pol I and III, and RNA Pol II termination?
Pol I and III transcripts have discrete 3' ends that are generated by cleavage, though mechanism for termination differs; Pol II transcripts have variable 3' ends, termination occurs through coupled cleavage and polyadenylation
What are the main processes that prepare the mRNA transcript for translation?
7-methyl-G-cap at mRNA 5' end, polyadenylation at mRNA 3' end, splicing
What are some other forms of processing that an occur to RNA?
Group I splicing (nuclear rRNA of simpler eukaryotes), Group II splicing (mitochondrial and chloroplast RNA), tRNA splicing (catalyzed), trans-splicing, RNA editing
Where is RNA regulated?
at many stages in its life cycle
What are hnRNPs?
heterogeneous ribonucleoprotein particles, an association with nascent RNAs of nuclear proteins and other RNAs
What are the three main functions of hnRNP binding?
present more uniform substrate surfaces for processing prevent formation of secondary structures e.g. Watson-Crick base pairing), facilitate interaction with other processing components, contribute to transport and proper localization
What are the four main steps of RNA processing in the nucleus?
5' capping of primary RNA transcript, cleavage at poly(A) site via endonuclease, polyadenylation via Poly(A) polymerase (PAP) and ATP, RNA splicing of transcript to remove introns
What is the purpose of the 5' RNA cap?
facilitates splicing, stabilizes mRNA transcript (against 5' degradation), facilitates transport of mRNA to cytoplasm, recognition by ribosomes in translation as RNA destined for translation
How is the 5' cap added?
a GTP is added to the 5' end via a 5'-5' bond (enzyme: guanylyltransferase), first few nucleotides (including GTP at N#7) are methylated (enzyme Guanine-7-methyltransferase)
What is the difference between a primary transcript and a nascent transcript?
primary is the product of RNA Pol on its template-- not processed and therefore not functionally active; nascent transcript is in the process of being transcribed
How are nascent transcripts capped?
an enzyme associated with C-term domain of RNA Pol II (phosphohydrolase) removes a gamma phosphate from the 5' end of the nascent transcript, guanylyltransferase adds a GTP and removes PPi, guanine-7-methyltransferase adds a methyl to the 7th nucelotides of nascent transcript, 2'-O-methyltransferase methylates the first 1-2 nucleotides of transcript
What residues are methylated in all caps?
the free N on the five-member ring of the capping guanine residue
What residues are methylated in cap 1?
the 2' O of the first residue
What residues are methylated in cap 2?
the 2' O of the second residue
What are the two separate methylation steps in 5' capping?
7' methylguanylate and then the first 1-2 nucleotides of the transcript
What RNA Pol enzyme is the 5' capping enzyme specific for?
What is polyadenylation?
addition of 40-250 untemplated adenosine nucleotides to the 3' end of the mRNA transcript
What does polyadenylation do?
contributes to mRNA stability, facilitates transport of mRNA, contributes to formation of a translation complex
What are the main factors involved in RNA polyadenylation?
CPSF binds AAUAAA on RNA, CstF bind U/GU-rich sequence on RNA, CFI and CFII connect both regions and loop them together, PAP adds As, PRPs bind the new poly-A tail
What process is coupled to pollyadenylation?
3' cleavage
Where are polyadenylation sites located?
internal to transcripts-- two main sites are necessary: AAUAAA site and GU/U rich site
What two signals are necessary for polyadenylation?
AAUAAA site (10-35 nucleotides upstream of poly-A tail) and GU/U-rich site, within 50 nucleotides of cleavage site
What happens to mRNAs that aren't polyadenylated?
they don't get processed, and unprocessed mRNA-->degraded
What is the frist step of polyadenylation?
4 factor CPSF forms an unstable complex with the aU site at the most 5' sequence, 3 more cleavage factors associate and their interaction with the GU/U-rich sequence stabilizes the complex
What enzyme polyadenylates mRNAs?
Poly-A polymerase (PAP), binds 10-35 nucleotides downstream of AAUAAA site
What are the two phases of polyadenylation?
slow addition of 1-12 bases, then rapid addition of 200-250 bases (aided by PABII)
What is PABII?
poly-A binding proteins, enhance rate of poly-A addition
What transcripts are polyadenylated and what ones are not?
histone proteins are not, also poly-A tail is longer in mammalian transcripts than in yeast
What is splicing?
the process by which introns (intervening sequences) are cleaved out of RNA and exons (expressed sequences) are fused back together)
What RNA processing event is an important source of protein diversity?
How was splicing discovered?
it was noted that DNA-mRNA heteroduplexes did not hybridize equally-- more DNA
Lecture 17
RNA Processing
how were junctional sequences (splice sites) identified?
comparison of cDNA and DNA sequences
What are the three most conserved splice sites?
5' splice site with GU, branch point with A, 3' splice site with AG; also a ~15 bp pyrimidine-rich region which can contribute to splicing
What experiment proved that splice sites are conserved?
the 5' SV-40 beta-globin sequence inserted intoTC cells could generate a chimeric gene that could be spliced
What is the mechanism of RNA splicing?
transesterification with lariat intermediates
What are the key regulatory residues of mRNA splicing?
5' end of intron, 3' end of intron, branch point in context of pyrimidine rich region
What energetic molecule drives splicing?
ATP-- however, the chemistry of the splicing reaction does not consume energy
What is the sequence of events in RNA splicing?
2' OH of branch point A attacks phosphate of 3' end of exon 1, removing it; 3' OH (missing its phosphate) attacks phosphate of 5' downstream exon (releasing 3' OH of intron) to generate splice and release branched lariat molecule
Where does splicing happen?
the splicesome
What happens in the splicesome?
5 snRNPs assemble sequentially on the pre-mRNA, then rearrangement, catalysis and finally release
What the hell is a splicesome?
50-70 protein complex (especially snRNPs)that catalyzes mRNA splicing, comparable in size to a small ribosomal subunit
What are snRNPs?
small ribonuclear protein particles, 5 of them make up the splicesome-- ,made up of pre-mRNA, snRNAs, proteins
What happens in snRNP assembly?
proteins, snRNAs and pre-mRNA undergo assembly mediated by sequences in 5' and 3' regions of the intron, 5 U-rich snRNAs participate in association with 6-10 proteins, base-pairing is necessary between snRNAs and the pre-RNA
What is the stereotypic structure of snRNAs in the splicesome?
what interaction is necessary between snRNAs and the pre-mRNA for splicing to occur?
complementary base pairing
What is the Sm region in snRNA and how is it correlated with disease?
I don't know what it does, but patients with lupus have an increased frequency of having autoantibody to a number of splicing components
What is the order of (re-)arrangement in the splicesome?
U1 and U2 associate via base-pairing, trimer of U4, U5, U6 join; =splicesome; snRNA bp interaction rearranges splicesome (U1, U4 snRNPs released); catalytic core is U6, U2) completes first transesterification, rearranges and completes second, lariat intron is released, debranched (by a debranching enzyme), degraded, then other snRNPs are released
What snRNPs are the catalytic subunits of the splicesome?
U2, U6
How much RNA is removed during splicing?
about 95% of the RNA Pol II product
What happens to released intronic RNA?
debranching enzyme opens the lariat structure; then it is susceptible to degradation because of it's unprotected 5'pG and 3' A-OH ends-- primarily undergoes exonucleolytic attack by exosome
What does the exosome do?
macromolecular machine composed of 11 exonucleases and a RNA helicase; can exonucleolytically attack the linear intron (mainly by 3'->5' hydrolysis, also can process splicing and polyadenylation mistakes
How can RNA processing be regulated?
Pol II CTD provides a platform for RNA processing factors
What enzymes have been demonstrated to have physical assications with the CTD region of Pol II?
5' capping enzyme, polyadenylation factors, splicing factors (together with CTD phosphorylation, these facilitate elongation)
How long is the CTD?
friggin' long-- looks like it's 6 times the diameter of Pol II in yeast, 12x in humans!
How big are introns/exons?
humans: av. intron is 3.5 kb (some are as long as 500kb), average exon is 150 bp
What do SR proteins do?
bind to RNA and recognize sequences in the intron to aid in splicing (interact with ESEs to mediate U1 and U2 snRNP homing)
What domains in SR proteins facilitate their activity?
RRM binding domains bind specifically do RNA and Ser/Arg interaction domains recognize sequences within the intron in the contex of the nearby exonic sequence, then specify the interaction of the protein with exonic splicing enhancers
What are ESEs?
exonic splicing enhancers, regions of mRNA that interact with SR proteins to recognize splice sites and with U1 and U2 to initiate splicesome
What is a cross-exon recognition complex?
complex of SR proteins with ESEs, that are bound to U1 and U2 to assist in snRNP homing
What is alternative splicing?
process by which multiple combinations of splice acceptor and splice donor sites are used in splicing an RNA transcript in order to generate multiple different lenghts and sequences of mRNA
What is a viral example of alternative splicing?
SV40 uses alternative splicing to generate the small and large T antigens, Adenovirus uses alternative splicing to generate multiple transcripts of the major late promoter (MLP)
How does alternative splicing generate the small t and large T antigens?
one splice form removes an intron but maintains an early stop codon (small t), one splice form removes intron and early stop codons, resulting in larger protein (Large T)
How is does adenovirus use alternative splicing to generate multiple gene products?
alternative polyadenylation leads to five potential termination products, alternative splicing can generate even more diversity between five exons
What are examples of splicing regulation in eukaryotes?
tissue-specific splicing (fibronectin), sexual differentiation in drosophila, C. elegans, neuronal-specific splicing
How is alternative splicing generally regulated?
RNA-binding proteins that bind specifically near regulated splcing sites (may act both positively and negatively)
What are Group I and Group II splicing?
autocatalytic splicing (self-splicing) mechanisms, yield less diversity than eukaryotic nuclear mRNA splicing, but provide evolutionary insight (found in limited number of transcripts)
What types of proteins undergo Group I splicing?
nuclear rRNA genes of protozoans
What types of proteins undergo Group II splicing?
protein coding genes and some rRNA and tRNA genes of mitochondria, and chloroplasts in plants and fungi
How do group I introns splice?
free soluble G makes a nucleophilic attack on the exon-intron junction (caps 3' of intron) free 3' OH of exon 1 attacks 5' end of exon 2, exon 1 and 2 are ligated via reformation of phoshpodiester bond
How do group II introns splice?
group II introns have conserved, complex secondary structure (similar to splicesome) two trans-esterification reactions, maturase proteins provide catalysis
What are the possible evolutionary roots of snRNAs?
group II-like RNA precursors-- could have continued to facilitate catalysis of splicing in trans-esterification rxns while introns evolved allowing greater shuffling/diversity
What are the products of catalytic RNA-mediated RNA processing?
group I yields linear product, group II yields branched product
What is a ribozyme?
a piece of catalytic RNA
What types of processing happen to pre-tRNAs?
5' extension (RNAse P), 3' UU conversion to CCA, base modification (extensive methylation), splicing
What is RNAse P?
RNAse P=polypeptide + M1 RNA; extends 5' tRNAs (RNA can do cleavage itself)
How does pre-tRNA splicing work?
distinct mechansims from mRNA processing or self-splicing, include one-step excision rxn, exonuclease and then activation of 3' and 5' ends of exons, religation
What are some essential features of pre-tRNA splicing?
one-step excision, ATP, GTP requirements, different intermediates, secondary structure is important, catalysis by proteins
What is trans-splicing?
140 nt leader RNA (from tandem transcription units) undergoes 2-step reaction to yield 5' 39 nt mini-exon, spliced to 5' end of protein coding regions from primary transcripts, triggering cleavage and polyadenylation
How are mobile introns made?
mobile introns are like trasposons that use group II machinery but encode reverse transcriptases and DNA endonucleases to insert themselves in random places in the mitochondrial genome
Lecture 18
Introduction to RNA Editing
When does RNA editing occur?
in situations where the sequence of mRNA is different than the coding strand, but not due to splicing events, whatever.
What machinery is necessary for RNA editing?
terminal uridyl transferase (TUTase), endonuclease, exonuclease, ligase (mammals also use specialized deaminases), gRNAs
Where is RNA editing common?
mitochondria of protozoans and plants and chloroplasts
What's an example of a protein that undergoes RNA editing?
Apo B-- made in liver and intestine (shuttles cholesterol); single gene produces mRNA that's processed differently in intestine (Apo B-48) and liver (intestine-specific deamination of mRNA at bp 6666 CAA->>UAA causing premature stop codon to produce Apo B-100)
How does RNA editing in mitochondria contribute to kinetoplast diversity?
RNA editing leads to extensive insertion or deletion of U residues, insertion can be so extensive it can double transcript lenth
How does kinetoplast RNA editing work?
guide RNAs from distal regions of kinetoplast genome hybridize to anchor sequences of pre-mRNA, editing starts near 3' end of genome and occurs in successive iterative phases
What are gRNAs?
guide RNAs, facilitate enzymes for RNA editing (drastic changes); transcribed from different region but complimentary to RNAs that will be edited
What are ribozymes?
ribonucleic acid entities with secondary structure that allows them to hybridize complementary RNA and use catalytic activity to cleave and inactivate target RNA
Where were ribozymes originally isolated?
plant viroids (ribonucleic entities that infect/harm plants)-- recognized that some RNA genomes underwent self-cleavage
How can ribozymes be used to cleave RNA
secondary structure of ribozymes can be used to make hammerheads (3 stem loops and conserved bases)-- have catalytic site in stem loop 1 and a cleavage site near the 5' end;
How might ribozymes be a potential therapeutic machine?
complementary base pairing can be used to target ribozymes, allowing cleavage of undesirable RNAs
What are some possible targets of ribozymes?
diseases that are genetically dormant (retinitis pigmentosa, ALS) cancer, HIV
What is ALS (AKA Lou Gherig's disease)?
dominant mutation in SOD1 gene leads to defective protein that accumulates as amyloid aggregates in motor neurons; degeneration of MNs leads to muscular atrophy, weakness, uncontrollable muscle spasms and twitching; onset during middle age and usually leads to death within 2-5 years
What is an aptamer?
chemically synthesized oligonucleotide with highly specific 3-D conformation, mimic or interfere with process of theraputic value
What is AMD?
progressive degenerative disease resulting in loss of central vision (causes severe vision loss of patients > 50; angiogenesis of new blood vessels, leak and cause scarring and vision loss; associated with elevated VEGF levels in eye
How are aptamers used to treat AMD?
block VEGF
How is RNA editing important in Ebola virus?
Ebola is ssRNA virus w/ 19 kb genome; glycoprotein on surface of virus is related to cytotoxicity; if virus is edited, nonstructural glycoprotein secreted, leading to enhanced cell death, if virus is unedited membrane glycoprotein is not secreted, leads to less cell death
What is required for translation?
adequate pools of AAs, mature processed mRNA in cytoplasm, tRNAs (charged with AA), ribosomes/polyribosomes, GTP and ATP
What are the three main roles of RNA?
tRNA: decoding mRNA and bring in proper AAs, mRNA: coding the nucleotide sequence, rRNA: part of ribosome, catalyzes reactions
What is a codon?
trinucleotide sequence within the mRNA that codes for an amino acid
what is an anticodon?
trinucleotide sequence in tRNA that correlates to a codon
what is an open reading frame?
reading frame of 3 nucleotides that will code for the correct protein
What can cause a framshift?
deletion or insertion of nucleotides
What is wobble?
a phenomenon that allows the 5' position of the anticodon to hybridize to several different nucleotides
what is translational frameshifting?
ribosome stops/stalls and therefore reads 4 nucleotides as 1 codon, ribosome backs up and therefore reads a nucleotide in more than one codon
How do you read a codon table?
vertical choice is first position, horizontal choice is second position, list of last position
How did Nirenberg derive the genetic code?
bound trinucleotide code and tRNAs and ribosomes to filter paper, see what tRNA-AA combo bound
What is the process by which an AA attaches to a proper tRNA?
charging or aminoacylation
What enzyme catalyzes aminoacylation?
amino-acyl-tRNA synthetase (a separate enzyme exists for each amino acid)
What is the product of tRNA charging?
a tRNA attached to an AA on its 3' end-- CCA is linker in tRNA sequence
What are the two types of adaptors necessary for decoding mRNA?
tRNAs and aminoacyl-tRNA-synthetases
What are the two main functions of tRNA?
chemical link to specific AA, base pair to mRNA codon
What bond in tRNAs facilitates translation?
high energy ester bond between AA and tRNA
How does the aminoacyl-tRNA-synthetase choose the right AA-tRNA pair?
it undergoes conformational change (fast with the correct pairing, slow with incorrect)
How does the aminoacyl-tRNA-synthetase proofread?
if incorrect pairing occurs, synthetase hydrolyzes ATP and removes AA-AMP and charged tRNA
What is the two-dimensional structure of tRNA?
clover leaf (characteristic stems and loops)
What loops exist in tRNA?
D loop (dihydrouridine in loop), anticodon loop (where decoding, base pairing occurs), variable loop, TpsiCG loop (pseudouridine), acceptor stem
What modifications happen to the base pairs in tRNA?
lots, making things like dihydrouracil, inosine, thymine, pseudouridine, methylations
What nts are common/uncommon in wobble?
A is rare, I (deamination product inosine) is common (e.g. 4/6 of LEU synonymous codons are read by 3'GAI)
What are four common non-standard (wobble) base pairings?
Uridine-Guanine, Cytidine-Inosine, Adenine-Inosine, Uridine-Inosine
Lecture 19
What RNA sub-type is an essential component of ribosomes?
rRNA! 60% of ribosomal mass in prokaryotes (less in eukaryotes)
What are the subunits of ribosomes?
large and small subunits
What are the three main roles of rRNA?
structure, assembly, catalysis
Does rRNA have a poorly- or well-defined structure?
well defined! Chemical cross-linking, photo cross-linking, enzymateic probing; significant secondary structure and base pairings between nts that are often quite distant-- secondary structure quite conserved across many species
What are the main processes in translation?
initiation (regulated assembly of ribosomes), elongation (polypeptide chain elongation), termination (reading stop)
What is necessary for initiation of translation?
regulated assembly of ribosomal subunits, activated tRNA(i)Met and mRNA, eIFs
What are the main processes involved in translation initiation?
regulated assembly of ribosome with activated tRNA(met) and mRNA, and scanning to find start sequence
What are eukaryotic initiation factors?
eIFs ensure that subunits don't assemble inappropriately-- EIF3 binds 40S subunit and eIF6 binds 60S subunit
How is the pre-initiation complex formed?
40S subunit binds eIF3, then eIF1A and then eIF2-GTP + Met-tRNA(i)Met
How does the pre-initiation complex become the initiation complex?
binding of eIF4 and mRNA
What is eIF4?
a cap-binding complex, composed of 4 polypeptides that binds to the 5' cap (through the 4E polypeptide) to associate with mRNA
What does the initiation complex do?
undergoes secondsary structure unwiding, scanning and start site recognition of mRNA (has associated helicase that melts out secondary structure)
What subunits make up the initiation complex?
40S, eIF1A, eIF3, eIF4(ABGE), Met-2-GTP-tRNA
How is intitation of translation begun?
initiation complex hyrolyzes ATP-->ADP, releases eIF1A, eIF3, eIF4 complex, eIF2-GDP + Pi (last is irreversible), 60S subunit binds with eIF6, eIF5-GTP, then eIF6 and eIF5-GDP are released
What does the 60S bring to the initiation complex?
his friends, eIF6 and eIF5-GTP
What is the complete ribosome?
80S, made up of bound 60S and 40S subunits
Where is the mRNA start sequence located?
100 nt downstream of 5' cap
What processes are involved in peptide elongation?
entry of succeeding aminoacyl-tRNAs, bond formation (peptidyltransferase), translocation of ribosomes, GTP hydrolysis
What is the first step of elongation?
tRNA charged and associated with EF1alpha-GTP will read A site, if tRNA is correct, GTP hydrolysis occurs, ribosome udergoes conformational change
What catalyzes the peptide bond formation between the Met and the A site AA?
rRNA of large subunit
How does the ribosome move down the mRNA?
binding of EF2-GTP and release of EF2-GDP and Pi
Peptide bond formation involves loss of what molecule?
How does the ribosome stop translation?
reading of stop signal, activation of termination release factors (GTP hyrdolysis) release and dissociation of the ribosome
What are termination release factors?
eRF1 (similar shape to tRNA, reads stop codon in A site), eRF3 bound to GTP
How do eRF1 and eRF3-GTP terminate transcription?
they block the A site, then tRNA is cleaved, then released, finally ribosome dissociates
What regulatory processes affect translation?
termination codons, initiation codons, initiator tRNA
What are termination codons?
What are initiation codons?
prokaryotes: GUG and AUG, eukaryotes just AUG
What is the difference between eukaryotic and prokaryotic initiator tRNA?
prokaryotes use f-met-tRNA, eukaryotes use met-tRNA
What two factors of prokaryotic translation allow for antibiotic specificities?
GUG start signal and f-met-tRNA initiation tRNA
What factors increase translational efficiency?
circularizing mRNAs, polyribosomes
How is mRNA circularized?
facilitated by initiation factors and PABP1 (physically associates with initiation factors and responsible for binding the 5' cap)
What do polyribosomes do?
allow for multiple copies of translation at the same time
What does circularization do?
allows dissociating ribosomes to start again quickly
What is the unique feature of prokaryotic initiator tRNA?
has formylated amino acid, therefore no base pairing between C-A (need three G-C base pairs to enter P site
What are aminoglycosides?
drugs that bind the 30S ribosomal subunit and prevent f-met-tRNA from binding the ribosome (e.g. kanamycin, neomycin, gentomycin, streptomycin)
What are inhibitors of translation elongation?
antibiotics that block prokayrotic elongation (eg. Kirromycin makes EF-Tu irreversibly bind ribosome, Fusidic acid makes EF-G-GDP irreversibly bind ribosome, Puromycin resembles charged tRNA and makes translation stop prematurely in prokaryotes and eukaryotes, chlormaphenicol inhibits peptidyl transferase in prokearyotes, cycloheximide inhibits peptidyl transferase in eukaryotes)
What is the mechanism of puromycin toxicity?
mimics 3' amino-acyl-tRNA, but blocks translocation of ribosome, stalls and dissociates
What do tetracyclines do?
block A site, inhibit binding
What does streptomycin do?
causes code misreading at low levels, inhibits initiation
What toxins affect eukaryotic translation?
diptheria-- inactivates eE2F, ricin-- 2 chain toxin, A chain depurinates 28S rRNA loop, irreversibly inactivating 60S subunit
What is an IRES?
internal ribosome entry site-- halts host translation while maintaining viral translation
What are viruses that use IRES?
polio virus, encephalomyocarditis virus, hepatitis C virus
What does an IRES do?
takes the place of the 5' cap
How can IRES be used in MBO?
can take the place of a promoter, can be placed between two genes to allow two genes to be translated from bicistronic mRNA
What are examples of viruses that disrupt host initiation machinery without affecting their own tanslation?
adenovirus and influenza-- eIF-4E is dephosphorylated and becomes inactive, poliovirus and encephalomyocarditis virus--eIF-4E is sequestered by a cell protein (4E-bp1) that is dephosphorylated in infected cells, polio virus and foot-and-mouth disease virus-- eIF-4G is cleaved by a viral protein
How do host cells limit initiation to limit viruses?
Normally eIF-2b recycles GDP on eIF-2a; but phosphorylated eIF-2a cannot be recycled--it binds to eIF-2b and GDP, inhibiting translation initiation
How do viruses circumvent eIF-2alpha inactivation?
adenovirus-- VA RNA activates Pkr (host protein that phosphorylates eIF-2alpha), HSV dephosphorylates eIF-2alpha, other viruses prevent host recognition of dsRNA so that eIF-2alpha is never phosphorylated, or block Pkr dimerization
Lecture 20
Gene Regulation
How can viruses cause frameshifting?
via slippage-- cause translation of alternative protein via same mRNA sequence
How do HIV and rous Sarcoma virus use frameshifting?
to translate the fusion peptide Gag-Pol; A-1 frameshift allows for reading of AA codon instead of stop codon
What does frameshifting accomplish for viruses?
allows for greater efficiency of genome
What common cell protein also involves frameshifting in its production?
How can mutations be supressed in translation?
nonsense suppressor: mRNA nonsense mutation is corrected by a mutation in anticodon that inserts and AA instead of terminating at that position; missense suppressor: inserts a proper AA by misinterpreting the mutant codon
What is amber suppression?
a stop codon is misread by the suppressor tRNA and the protein is extended past the usual endpoint
What is the major difference between prokaryotic and eukaryotic gene regulation?
prokaryotes always have basal transcription, so much regualtion is via repressors (also have multiple genes regulated by one operator); eukaryotic has minimal to no basal activity so main regulation is via STFs that bind enhancers (one gene per enhancer, or per mRNA)
How can mRNA stability serve as a regulatory mechanism?
half-life of mRNA contributes to level of translation product in a cell
What are some common eukaryotic transcriptional regulatory mechanisms?
transcription initiation (hormones), mRNA processing mechanisms (calcitonin/CGRP, IgM heavy chain), mRNA stability (transferrin receptor, ferritin), tranlational initiation (heme-containing proteins, growth factors), DNA rearrangements (trypanosome VSGs), regulation by RNAs
How do hydrophobic and hydrophilic hormones work?
hydrophobic (peptide) bind cell-surface receptors, hydrophyllic (cholesterol-derived) cross membrane and bind intracellular receptors
What are the two main families of intracellular hormone receptors?
cytosolic and nulcear
What is the nuclear receptor family of TFs?
includes glucocorticoid receptor (GR), estrogen receptor (ER), thyroid hormone receptor (TR), retinoic acid receptor (RAR), progesterone receptor (PR); general structure is: H2N-variable region-DNA binding domain-hormone binding domain-COOH
What are the domains of nuclear hormone receptors?
variable region (activating/repressing domains), DNA binding domain (DBD) binds HRE (hormone response element), ligand binding domain
How does the glucocorticoid receptor work?
when no hormone present, LBD of GR has Hsp90 bound, upon hormone binding, receptor undergoes conformational change and Hsp90 is displaced and hormone-receptor duplex can enter nucleus, DBD positively or negatively regulates transcription
Give two facts about glucocorticoids
get their name because they cause an increase in glucose levels in blood, cortisol is a glucocorticoid that suppresses immune response and inflammation
How does retinoic acid signal in the cell?
RAR and RXR (2 RA receptors) can dimerize with each other in presence or absence of RA (and various other hormones they interact with)-- decreased RA increases RAR-RXR dimer, binds HRE, activates NCoR (co-repressor), activates HDAC and deacetylates histones; increased RA causes RAR-RXR-RA, binds enhancer, activates co-activators and activates transcription
How can thyroid hormone act through RXR?
RXR can dimerize with thyroid hormone receptor (TR)-- in absence of thyroid hormone, RXR-TR activates TRE, activates co-repressor, causes histone deacetylation and inactivation of genes; in presence of thyroid hormone, RXR-TR-TH activates enhancer, activates co-activators, causes histone acetylation and actiavtion of genes
What are transcriptional co-repressors and co-activators?
histone deacetylases (mRPD3, Sin3, NCoR, RXR-TR complex) and histone acetylases (p300/CBP, TH-RXR-TR complex)
How can a patient develop tolerance to steroid drugs?
if a hormone that activates histone acyl transferase is given for a long time or at high doses, eventually it causes the acetylation of proteins like transcription factors, causing them to fall off (inhibiting transcription)
Lecture 21
Gene Regulation
How can poly-adenylation lead to gene regulation?
many pre-mRNAs can be processed to contain one of multiple poly-A sites and/or splice patterns, can lead to mRNAs of different sequences, stabilities and lengths
What are three ways that multiple poly-A sites can cause different gene products?
tandem poly-A sites within same gene, composite exons (fuse a portion of one exon to another), skipped exons (multiple 3' exons possible)
What are two examples of how differential polyadenylation can affect expression of gene products?
calcitonin/CGRP expression and IgM heavy chain alternative processing
How is the calcitonin/CGRP gene regulated differently in tissues?
thyroid generates calcitonin-- involved in regulation of Ca levels, tissue; neurons generate Calcitonin Gene Related Product (CGRP), neuropeptide involved in cardiovascular homeostasis
How do thyroid cells specify calcitonin expression?
tissue-specific splicing factors (PTB and SRp20) stabilize the splice site at the 4th exon-- gene is spliced and polyadenylated at the 4th exon
How do neurons specify CGRP expression?
absence of splicing factors lead to splicing to exons 5 and 6
What are the exclusion/inclusion factors for calcitonin/CGRP expression?
exclusion: weak 3' splice site, weak core p-A signal (poor DSE), limiting PTB and SRp20 in neuronal cells, repressor in neuronal cells; inclusion: intron p-A enhancer and sufficient PTB and SRp20, two exonic splicing enhancers, stem-loop containing 3' splice site and first part of exon, possible positive effector acting in first part of exon
How is IgM heavy chain alternative processing important?
determines soluble vs. membrane-bound protein
How is IgM heavy chain processing regulated?
different poly-A sites allow for multiple mRNA forms
What's the most common mechanism for mRNA degradation?
gradual erosion of the poly-A tail-- when PABPI can't bind, loose interactions with cap and eIFs, net result is decapping (note: higher translation leads to greater poly-A stability)
what regulates mRNA half-life?
lots of things-- anything that regulates deadenylation or exosome degradation…
How is beta-globin mRNA stability regulated?
in presence of GMCSF mRNA half-life is very short
What repeat is involved in the GMCSF-mediated shortening of beta-globin mRNA half-life?
AUUA repeat-- beta-globin without AUUA has longer half-life
What are examples of extracellular signals that affect mRNA half-life?
prolactin-induced casein mRNA stabilization, small molecule destabilization (transferrin-iron receptor TfR)
How does iron regulate transferrin mRNA half-life?
transferrin receptor (TfR) brings in iron from blood when iron is low; TfR mRNA has IRE (iron response element) region at 3' end with secondary structures rich in AUs that must be bound by IRE-BPs to prevent mRNA degradation; decrease in Fe leads to increase in IRE-BP, increases TfR mRNA half-life, increases iron uptake
What are two ways that IRE-BP contributes to translational regulation?
by binding and blocking AU-mediated degradation (transferrin), and by binding and blocking IRE-based blockage of ribosomal scanning/binding (ferritin)
What does mRNA surveillance do?
avoids translation resulting in abnormal proteins: exosome function, nuclear export, nonsense-mediated decay
How does the exosome contribute to mRNA surveillance?
recognizes and degrates mRNAs with splicing and poly-A errors (both cytoplasmic and nuclear)
How does nuclear export contribute to mRNA surveillance?
restricted for incompletely spliced mRNAs and splicesome-associated mRNAs; others can't get out
how does nonsense-mediated decay contribute to mRNA surveillance?
mechanism to degrade mRNAs occurring with stop codons before last splice junction in mRNA; exon junction complexes remain associated and trigger delay… yeah. They're still workin' it out.
How does heme presence regulate translation of heme-containing proteins?
absence or low heme activates HCR (heme controlled repressor) which phosphorylates eIF2-a, preventing recycling and blocking translation
How can growth factors regulate translation?
cell surface receptor binding can trigger receptor tyrosine kinase (TRK), activating mTOR, activating PHAS-1(P), activating eIF-4E to initiate tranlation
How can PHAS-1 regulate translation?
dephosphorylated PHAS-1 sequesters eIF-4E, phosphorylation (by activated mTOR) allows separation, eIF-4E can bind eIF-4G and activate capping to initiate translation
How can chromosomal position changes regulate gene activateion?
expression from telomeric sites allows transposition to ES with unusual flanking sequences, transcribed by Pol I in extranucleolar body
How can trypanosomes avoid immune detection?
change surface coats presented to host-- they move the location of the basic copy of the gene for one of 10e7 different surface glycoproteins (VSG) into expression sites
What are micro RNAs?
short RNAs that hybridize to 3' UTRs of target genes and cause translational repression
How does miRNA or siRNA work?
dicer exonuclease cleaves dsRNA (loop or straight) to ssRNA, associates with RISC complex, perfect bp causes target cleavage, incomplete bp causes translation repression
What might be an endogenous use of RNAi?
elimination of abundant (defective?) mRNAs, potential defense against transposons and viruses
Lecture 22
Retroviruses and HIV
What are negative and positive RNA?
viral RNA that is either translated (+) or convertd to positive RNA and translated (-)
How do retroviruses produce protein?
through a DNA intermediate
What is the RNA-dependent DNA Polymerase used by retroviruses?
reverse transcriptase
What happens to the duplex DNA product of RT?
integrated into host genome (by integrases)
What is a provirus?
integrated version of viral genome
What is the lifecycle of a retrovirus?
RNA duplex and RT and integrase in viral particle, enter cell, RT copies to DNA, integrase integrates it into host DNA, host RNA pol transcribes, host machinery generates diploid RNA and RT and integrase, packaged into viral particle
What is different between viral mRNA and genomic RNA?
mRNA has 5' capping and 3' poly-A
How do retroviruses increase efficiency in their genome?
alternative splicing, frameshifts, proteolytic processing (each gene in the viral genome encodes multiple proein products)
How do viruses leave/join host cells?
RNA genome is packed into a core surrounded by capsid proteins, particle pinches off from host membrane, maturation continues after release, infection is essentially the reverse of process
How does viral DNA change during infection/integration?
RNA genome has short direct repeats on both ends (some variation in size) and untranslated U5 and U3 (of various size); linear DNA has long terminal repeats (identical at 5' and 3' but different from genomic ends); provirus has loss of 2bp at each end of LTR
What important features does the U3 region of the LTRs contain for viruses?
promoters and enhancers important for expression of RT
How is retroviral DNA generated?
uncharged host tRNA binds primer binding site (PBS), DNA synth starts until ~100-200bp from end (product jumps to become new primer), tRNA primer is removed, RNAse H degrades much of original RNA tmplate strand (fragments serve as primers) another jump adds U3 and U5, 5'-->3' synthesis completed
How is template switching important in retrovirus reverse transcription?
generates multiple primers from tRNA primer, also allows U3 and U5 addition (symmetric)
How does viral integrase catalyze DNA insertion?
converts blunt ends into recessed ends (2bp recessed 3' ends in LTRs), makes staggered cuts in target DNA (usually not site-specific), links recessed 3' ends of LTRs with staggered 5' ends of target DNA, existing gaps can be repaired by host enzymes (note integrase holds on to viral genome to facilitate integration)
How can retroviruses cause cancer?
virus containing oncogene, or viral promoter activates oncogene, also can integrate into middle of tumor suppresor gene and inactivate it
What's an example of an oncogenic virus?
Rous sarcoma virus (RSV)
How do transforming (transducing) oncogenic viruses work?
acquired host sequences by (non-homologous) recombination, are replication defective (dependent on helper virus) but recombination by wt virus allows transcription, splicing and packaging of defective genome; if recombination incorporated cellular proto-oncogenes, incorporation into host DNA or mutation can render these genes oncogenic
What's an example of a viral oncogene?
v-ras, mutations cause lack of regulation, constitutive activation of ERK MAPK pathway (GTP-bound Ras is constantly active)
What's an example of a gene activated by nearby integration of viral genome?
c-myc (also c-mos, c-H-ras)
What cells does HIV target?
CD4+ TH cells
What is the virion structure of HIV?
genome with nucleoprotein, RT, integrase and protease, surrounded by capsid, surrounded by envelope (membrane) with TM and receptor-binding proteins
What proteins make up HIV?
structural proteins (gag): MA-matrix, CA-cupid, NC-nucleocaspid, P6; envelope proteins (env): SU (gp 120), TM (gp 1); enzymes (gag-pol): PR (protease), RT (reverse transcriptase), IN (integrase); accesory proteins: Vpr, Vif, Vpu, Tat, Rev, Nef; also has uncharged host tRNA for priming and APOBEC3G, another host protein
What's the HIV life cycle?
uncoating of viral particle, activation of RT in cytoplasm, replicated DNA translocates to nucleus (Vpr helps), integrates, transcription of integrated genome (Tat helps: anti-termination factor), RNA exported as genome
How does HIV inhibit the activation of cytidine deaminase?
through Vif blockage
Why would cytidine deaminase activity be bad for HIV?
APOBEC3G makes C>U changes that become G>U mutations after + strand synthesis; diminishes HIV-1 replication at least 3 ways: host enzyme will recognize mispairing of proviral DNA (if glycosylates are active, viral genome will be destroyed), changes can result in extensive strand breakage (impairs synthesis), gives rise to G>A hypermutations which influence functioning of viral genome
What viral proteins facilitate nuclear import?
Vpr and MA (matrix protein)
What are important HIV transcription regions?
LTR: U3 contains promoter and enhancer, R contains start site of transcription and cleavage and poly-A site (multiple host transcription factors are used e.g. NFkB, TFIID, SP1)
How does Tat transcription stimulate more transcription?
original antitermination leads to elongation of transcript; as Tat protein is made, it facilitates CTD phospohrylation, leading to increased Tat production
What are three possible consequences of newly transcribed viral RNA?
unspliced undergoes Rev-mediated transport-->new viral genome; singly spliced undergoes Rev-mediated transport-->exits nucleus (requires RRE or Rev response element); multiply spliced undergoes Rev-independent transport
How does HIV stabilize infection?
Nef and Vpu change cell surface molecules: Nef causes CD4 and MHC I endocytosis (Rev-mediated endocytosis or RME); Vpu targets CD4 in ER to ubiquitin pathway
How are virions assembled?
packaging signal (psi) has an important role, 2 genome copies, structural proteins and gag-pol are packaged, virus buds off, taking portion of host membrane with it
What are the consequences of HIV infection?
indirect T cell death, long-term immunosuppression
What are the major anti-HIV drugs?
nucleoside analog reverse transcriptase inhibitors (NRTIs) disrupt viral DNA synthesis, protease inhibitors ihibit maturation of virus, non-nucleotide reverse transcription inhibitors (NNRTIs) block RT activity, fusion inhibitors block viral uptake, siRNA target viral mRNAs
What are the issues involved with HIV treatment?
side-effects, containment not cure, anatomical sanctuaries, high mutation rate, T cells must be restored
Why is it a challenge to make an AIDS vaccine?
HIV is a difficult target for neutralizing antibodies (conserved targets are concealed, exposed targets are variable, targets have glycosylation camouflage), HIV is able to form latent proviral DNA early in infection but with long half-life, HIV has high variability (>12 clinically relevant subtypes, inter-subtype recomination, high RT error rate)