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157 Cards in this Set
- Front
- Back
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What is the spontaneous mutation rate?
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1 per 10^9 bases per cell division
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What is the number of bases in the mammalian genome?
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>10^9
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How many mutations occur per cell division?
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1 per genome per cell division
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How many cell divisions occur per second?
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10^7
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Most of DNA is non-coding. Give a percentage for this information.
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less than 2% of human genome contains informative information
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Most mutations are what?
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inconsequential
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What happens to mutations that do cause a cellular defect?
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They will most likely kill the cell so they will typically not cause diseases such as cancer
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How many relevant targets for cancer?
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approximately 200 genes
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Average size of a gene (coding sequence, splice sites, and key regulatory regions)?
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approximately 2500-5000 bp
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What is the target size in the genome for mutations for cancer?
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approx. 5 x 10^5 bases.
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So 5 x 10^5 relevant bases/ 6 x10^9 total bases means that...
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each mutation per cell division only has a 0.008% chance of hitting a cancer gene
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What factors reduce the risk of mutations being dangerous?
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1.) many mutations are silent
2.) need at least 6 different alterations to cause cancer 3.) plus these mutations must occur in the same cell 4.) mutations that cause cancer cannot occur in a random order |
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What increases cancer risk?
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inherited mutations in tumor-suppressor genes
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Name four common features of DNA repair.
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1.) Must identify/recognize problem
2.) Must remove problem 3.) Must resynthesize corrected DNA 4.) Coordinated with Cell Cycle |
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DNA lesions that require repair. Name the cause of each lesion.
1.) missing base 2.) altered base 3.) incorrect base 4.) bulge 5.) linked pyrimidines 6.) strand breaks |
1.) acid, heat, glycosylases
2.) radiation, alkylating agents 3.) tautomers, proofreading defects 4.) intercalating agents 5.) UV irradiation 6.) ionizing radiation, chemical agents |
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Tautomers can cause spontaeous mutations. Name the tautomeric forms of the DNA bases.
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1.) adenine and cytosine - amino (common) and imino
2.) guanine and thymine - keto (common) and enol |
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Give the rare imino tautomeric form of cytosine. What changes about the cytosine in its imino form? What does this mispair result in?
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C-A / the location of hydrogen donor and acceptor groups / a transition mutation
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Tautomeric miss-pairings:
1.) A:T 2.) T:A 3.) C:G 4.) G:C |
1.) A (imino):C
2.) T (enol):G 3.) C (imino):A 4.) G (enol):T |
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What do chemical mutagens do to cytosine and adenine? What do these bind to?
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cytosine - uracil, adenine - hypoxanthine / uracil-adenine, hypoxanthine-cytosine
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What does UV irradiation do?
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breaks the hydrogen bonds in a pyrimidine (thymidine) dimer
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Name two types of DNA repair.
Name the steps of each. |
1.)base excision repair (deamination)
DNA glycosylase repair nucleases DNA polymerase beta DNA ligase 2.)nucleotide excision repair (dimer) repair nucleases DNA polymerase beta DNA ligase |
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Give six steps of nucleotide excision repair.
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1.) damage recognition (two pathways)
2.) DNA strand separation (XPB/XPD proteins) 3.) 3' XPG incision/5' XPF/ERCCI 4.) Excision 5.) DNA repair synthesis 6.) strand repair by ligase |
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What is MMR and when does it occur?
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mismatch repair / post-replication
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Give the four steps of MMR in bacteria.
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1.) normally, the parental DNA strands are methylated on certain bases, so
2.) mutations on the newly replicated strand are identified by scanning prior to methylation of the newly replicated DNA 3.) the mutations are repaired by excision repair mechanisms 4.) only after repair, are the newly replicated strand methylated |
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Name three general DNA repair pathways.
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Mutations that occur during DNA replication are repaired when
possible by proofreading by the DNA polymerases Mutations that are not repaired by proofreading are repaired by mismatch (post-replication) repair followed by excision repair Mutations that occur spontaneously any time are repaired byexcision repair (base excision or nucleotide excision) |
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What is a down side of DNA repair?
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It's hard to balance the need to evolve.
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What is a genome in terms of modern molecular biology?
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an organism's hereditary information encoded in DNA (or, for some viruses, RNA). The Genome includes both the genes and the non-coding sequences of the DNA.
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Give three details of the e. coli k-12 genome.
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1.) 4289 Open Reading FrameS
2.) 115 Structural RNAs 3.) 4.6X10^6 bp |
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Give four details of the human genome structure.
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1.) 23 pairs of chromosomes
2.) 3 GB 3.) ~50% repetitive 4.) only 3% protein coding |
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The majority of genes have......
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......no known function
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Name three 21st century techniques.
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genomics informatics
functional genomics microarrays proteomics |
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Describe the steps of microarray.
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extract mRNA and perform reverse transcription in order to label the mRNA from the two diffrent cells with two different fluorescent dyes (cDNA), combine the two in equal amounts, hybridize the target to the microarray and scan
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What does the transition from ds-ssDNA lead to?
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rise in UV absorbance
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T_m = ?
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50% melt point
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As T_m rises, ....
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genome complexity (thermal denaturation of DNA duplexes)% rises
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Which has a higher absorbency: single or double-stranded DNA?
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single-stranded
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What does Cot reflect?
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DNA concentration and time of incubation (amount of time it takes to go from single stranded to double stranded)
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What is important in re-association kinetics?
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size
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What do smaller genomes do faster and why?
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re-anneal because there are more copies (partners)/ug
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Define the difference between reannealing prokaryotic genomes vs. eukaryotic genomes.
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prokaryotic genomes Reanneal Smoothly Reflecting Largely Single Copy Genomes, Eukaryotic Genomes Reanneal at Different Rates-Reflecting DNA Components of Widely Different Concentrations
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Give the length of the following:
1.) satellite DNA 2.) minisatellite DNA 3.) microsatellite DNA |
1.) 5-X00s bp
2.) 1-100 bp 3.) 1-5 bp |
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Where is the human genomic structure highly repeated?
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around the centromeres and the telomers
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What does ethanol consumption do?
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induces expression of TCA enzymes
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What does extreme green represent in an ethanol consumption chart? Extreme red?
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>16X repressed / >16X induced
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After ethanol exposure, what is represented in 4-12 hours?
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where ethanol is high and glucose is low, resulting in high levels of induction (red)
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What type of RNA is found in prokaryotes? Where are their genomes found?
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multi-cistronic mRNA / in operons
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What part of an eukaryotic gene is removed?
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introns
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Name three things that complicate eukaryotic genes.
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1.) alternative splicing
2.) two poly A sites lead to choice of alternative 3' exons 3.) alternative promoters can lead to tissue specific mRNAs |
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What is RNA polymerase's responsibility?
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incorporating ribonucleoside triphosphates into RNA strands whose sequence is complementary to DNA template
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What does RNA polymerase need to bind to DNA at the promoter?
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transcription factors
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How does RNA polymerase move?
What is the other strand's function? |
along DNA template strand 3'-5'
it's coding because it has the same sequence as RNA |
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How does nascent DNA grow?
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5'-3'
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Give five characteristics of transcription.
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cyclical
dynamic highly regulated protein and DNA unchanged hydrolysis of PPi by pyrophosphatase releases a lot of energy and makes this rxn essentially irreversible |
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Give the five stages of the transcription cycle.
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1.) template binding
2.) chain initiation 3.) promoter clearance 4.) chain elongation 5.) chain termination and RNA polymerase release |
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What is the same as the template strand? What is the difference?
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Template DNA strand is the same as the built mRNA except for T to U and the deoxyribose/ribose story
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What is the cytoplasmic face of the nuclear pore?
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the peripheral ring structure
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What is the nuclear face of the nuclear pore?
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the inner ring basket structure
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How big is the nuclear pore complex, what type of symmetry does it have, what is it made of, and what does it mediate?
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huge (~30X ribosomes) / exhibits octagonal symmetry / 8 copies of ~30 highly conserved proteins known as nucleaparins / nuclear export and import
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Define bacterial RNA polymerase.
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5 subunits that can bind DNA RANDOMLY to transcribe RNA
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What does core RNA polymerase do without sigma factor?
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does not recognize and bind to specific DNA sequences (Initiation sites) within promoters.
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A single RNA polymerase has _____ as much force as a myosin motor.
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2X
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What does a sigma factor provide and when is it removed?
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specificity to polymerase / once transcription begins
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What does association with sigma factor form?
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complete haloenzyme with 5 Core subunits: 2alpha,1 beta, 1beta’,(1 omega not shown) to allow promoter binding and strand separation (transcription initiation)
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What are the six steps of mRNA export?
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1 & 2) the splicesome places the exon-junction complex 20-24 nts upstream of each exon/exon junction. aly is one ejc component
3) tap associates with ejc to drive it thru th npc (4) 5.)tap and aly are shed once mRNP is on cytoplasmic side 6.) remainder of ejc is shed uring first round of translation |
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What do bacterial transcription termination sequences cause?
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RNA formation of hairpin loops
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Name what is needed for transcription template for eukaryotic RNA polymerase II transcription.
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nuclear compartment
nucleoprotein complex----chromatin elucidation of the key cis-elements responsible for activation and repression of transcription (promoter elements and cis-regulatory elements) |
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Define the following:
TAFs TFIIB TFIIF TFIIH |
- TBP associated factors
- provides RNAPII binding site - subunit of which is related to sigma binds to entering RNAPII - has 9 subunits; 3 of which are catalytic: kinases / DNA helicases |
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What does a bead on a DNA strand allow us to monitor?
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the speed of transcription and allows us to find out the force that it can apply (how much force it takes to hold the bead in one position in order to prevent transcription)
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What are the two basic elements of a bacterial promoter?
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The -35 bp “upsteam” element
The -10bp ("Pribnow Box”) upstream element |
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What does alignment of multiple bacterial promoters show?
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Strong Consensus at -35 Element
and at -10 Element (Pribnow Box) |
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How many sigma factors are in e. coli?
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5
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What are the two types of bacterial transcription termination? What does the first require?
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Rho Independent (requires termination sequence) and Rho Dependent
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Does bacteria have exons?
Does bacteria have introns? |
yes
no |
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What do introns allow us to do?
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to make more copies than usual and to gain more time control
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Give another name for primary RNA.
How many transcripts go to mRNA? |
hnRNA / 1
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What is different about eukaryotic transcription? What controls this process? Name stages.
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it's much more complex and tissue specific / relationship between chromatin and RNA polymerase / tight=silent, loose=active
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Name the type of RNA synthesized by RNAP I, II, III, and IV.
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I = larger rRNAs
II = mRNAs, most small nuclear RNAs, microRNAs, and telomerase RNA III = small RNAs, including tRNAs, 5S rRNA, and U6 snRNA IV = (plants only) siRNAs |
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Where do cis-elements exist?
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on the same chromosome as the coding sequence
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What does the TATA box act as?
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a landing site for transcription complexes
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What are TBP's?
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TATA binding proteins
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Where does the TATA box exist?
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in eukaryotes and prokaryotes
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What brings in transcription factors?
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TATA binding proteins
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What has a heptapeptide (7aa) repeat?
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RNA PII CTD
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What is Ser5?
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the site for TFIIH-based CTD Phoshorylation that is required for RNAPII Activity
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What are EL/SII and PTEFb?
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ELONGATION Factors Req’d for Processivity (Ser2-P) by PTEFb
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What happens to the RNAPII tail when an inactive pre-initiation complex turns into an active initiation complex?
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it's phophorylated by the kinase activity
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Give the four steps of the formation of basal transcription initiation complexes.
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1.) Binding of TFIID to TATA is mediated by TBP binding
2.) TFIIB and TFIIH bind to complex where TFIIB recruitment is the rate limiting step in the formation of transcriptional initation complex 3.) RNA Pol II,TFIIB,TFIIE, TFIIB are linked to TFIID complex by PolII CTD 4.) CTD phosphorylated by TFIIH and is released by the complex to initiate Transcription |
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What is the function of the TBP associated factors?
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Stablize TBP complex by linking to upstream enhancer binder proteins
(Activators or TFs) which raises the Transcription from Basal to Activated Levels |
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Name three multi-functional proteins with specific domains that can act independently.
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- DNA binding (to both promoters and enhancers)
- TransActivation (to facilitate activation of transcription) - Protein-Protein Interactions |
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Define the homeobox DNA binding domain.
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- Homeotic genes (Drosophila) and Hox gene (mammals)
- The Homeobox Domain is a HIGHLY CONSERVED Stretch of 60aa that Mediates DNA Binding |
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Define bHLH proteins.
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MyoD
Achaete daughterless Obligate Dimers that have both DNA and Protein Binding Domains |
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What comes from very tight protein-protein connection?
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leucine zipper protein interaction with DNA
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What is the function of modular transcriptional enhancers?
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Bind Multiple TFs that often act in Combinatorial Manners
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What influences the level of gene expression?
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Protein-Protein Interactions between chromatin and Transcription Factors (TFs)
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What do TF's mediate?
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methylation, acetylation and phosphorylation of chromatin in a process known as remodeling
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What do activators (aka: transcription factors) bind to? What forms activated transcription?
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Activators bind to enhancers, dna folds onto tata to form activated transcription
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Give the charge of DNA.
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negative
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What is a zinc finger?
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Another transcription factor
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What do transcription factors bind?
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promoters or enhancer regions that are usually upstream of promoters
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What is chromatin remodeling?
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changes in chromatin structure, i.e. the shifting of nucleosome arrangement along the DNA
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Give the four steps of how TF binding to enhancers/promoters alters chromatin.
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1.) DNA is wound around a histone core, forming nucleosomes
2.) initial TF binds to a nucleosome core, displacing part of the histone core 3.) Additiona factors may bind the complex, further destabilizing the histone core 4.) When the histones have been displaced, other TF's may bind |
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What does TF-associated histone acetyltransferase do?
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Modifies Tails of Histone to Alter Their Conformation with Nucleosomal DNA-this
“Loosens/Opens” up the Chromatin to allow TF/RNAP Better Access |
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Modification of Histone tail residues:
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Methylation (N-LYS)
Acetylation of N-terminal LYS Phosphorylation (SER) To reduce overall POSITIVE charge in order to get the negatively charged DNA apart from it |
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Why would distant enhancers be brought closer to promoters?
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in order to modify the activity of the basal apparatus by folding the DNA
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Name five polyadenylation events.
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- CPSF = cleavage and polyadenylation specificity factor (recognizes AAUAAA)
- CstF = Cleavage Stimulating Factor - CFI/CFII = Cleavage Factors - PABP = PolyA Binding Protein - PAP = Poly A Polymerase |
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When does mRNA splicing occur?
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after 5'/3' modifications
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What is the next step in eukaryotes after transcription?
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transport of mature mRNA's out of the nucleus for cytoplasmic translation (known as mRNA export) - this is a critical difference between eukaryotes and prokaryotes
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Name seven characteristics of the genetic code.
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- universal (not quite, mitochondrial code uses different codons)
- read 5'-3' - nonoverlapping - unambiguous (1 codon = 1 aa) - degenerate (more than 1 codon/aa) - 3 stop codons (UAA, UAG, UGA) - 1 start codon (AUG) |
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What do similarities between aa's allow for?
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fewer mistakes/mutations
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What does the anticodon do?
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ensures that the correct aa gets placed in the sequence (recognizes the mRNA codon)
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What was Crick's wobble hypothesis?
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the same tRNA can recognize more than one codon
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What allows for some latitude at the 5' end of the anti-codon loop?
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The steric requirements at the anti-codon/codon recognition
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What two things can uridine bind with?
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guanine and inosine
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What three things can inosine bind with?
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cytidine, adenosine and uridine
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Give the three phases of translation.
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- initiation
- elongation - termination |
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Give the four components required for translation.
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1. RNAs: mRNA, rRNA, tRNA
2. Amino acids: 20 amino acids attached to tRNAs 3. Ribosomes: small subunit and large subunit 4. Auxiliary protein factors |
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Define tRNA charging.
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20 Distinct Aminoacyl-tRNA Synthetases Recognize Specific BPs /3D structure/anticodon of tRNAs to Correctly Place the Appropriate AA
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What do ribosomal RNA display?
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complex secondary structures
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How is ribosomal RNA transcribed?
What are the sites of ribosome synthesis? |
- off templates of rDNA multi-gene clusters in Nucleoli by RNAPI/III
- nucleoli |
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What are rRNA's post-transcriptionally modified by?
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High % Methylation of
Ribose and Conversion U ->Psuedo U |
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What does the processing of rRNA's occur through?
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EndoNuclease Cleavage
Mediated by small nucleolar RNPs (snoRNPs) |
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Name the subunits and the assembled ribosomes of prokaryotes and eukaryotes.
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pro: 50S, 30S / 70S
eu: 60S, 40S / 80S |
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Give the three ribosomal nomenclatures and their functions.
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- aminoacyl: beginning where RNA binds
- peptidyl: where aa chains are built - exit: end where RNA breaks off |
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Name some prokaryotic initiation factors.
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initiator tRNA with formyl-Met
IF1 IF2 IF3 |
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Name the difference between methionine and N-Formylmethionine.
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methionine has NH3 coming off its fourth side whereas N-Formylmethionine has an N which has an H and a C and the C has an H and a double bonded O
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Where should the 30S be positioned?
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the start site
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What is required for prokaryotic translation initiation? Which are lost? What is hydrolized and why?
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IF's 1-3 / GTP / to release IF2-GDP and 50S subunit binds
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What is the function of IF1?
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aids attachment to 30S
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What is the function of IF2?
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required for attachment of 1st aa-tRNA known as the initiator aa-tRNA to the P-site
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What is the function of IF3?
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blocks 50S entry
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Give two factors of the shine-delgarno sequence.
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- it's 5-10nt upstream (5’) of initiator AUG
- it is complimentary to sequence at 3’ end of 16S rRNA on small 30S ribosomal subunit |
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How is aa-tRNA added to A site?
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through complex with EF-Tu-GTP (Euk:eEF1alpha) and through complementary between codon/anticodon
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What does binding of A-site aa-tRNA cause?
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hydrolysis of EF-TuGTP->GDP
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What is regenerated for the next step though EF-T's?
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EF-Tu GTP
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What do P-sites and A-sites do while still binded to tRNA?
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form PEPTIDE BOND thru activity of Peptidyl Transferase component of 50S subunit rRNA (a ribozyme)
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What does amine N of A-site aa-tRNA do? What does this result in?
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carries out a nucleophilic attack on carbonyl carbon of P-site aa-tRNA which displaces the tRNA at the P-site / the A-site tRNA bound to a dipeptide
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What occurs during translocation?
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the ribosome moves 3 nucleotides (1 codon) in a 5’-3’ direction along the mRNA
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Where does the tRNA dipeptide move from? Where does the empty deacylated tRNA move to? What does this regulate?
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A->P site / E-site (EXIT) / EF-G-GTP ->GDP (eEF2 in Euks)
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What happens after deacylated tRNA leaves E-site?
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next aa-tRNA is
Free to enter the A-site with the help of EF-TU-GTP to start the process again |
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What does no aa-tRNA exist for? What does this halt?
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termination or stop codons / further elongation
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What are release factors required for?
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to recognize STOP codons (RF1:UAA/UAG; RF2: UAA/UGA)
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What does RF3-GTP work with, what is it hydrolyzed to, and what does this hydrolization do?
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RF3-GTP works with other RFs and is hydrolyzed to RF3-GDP to drive release
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What do release factors do?
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enter A-site, and drive release of nascent polypeptide from P-site tRNA
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Give the three steps of eukaryotic initiation.
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1. Scanning or Kozak >90% of all eukaryotic mRNA
2. Internal ribosome entry site (IRES) 3. Re-initiation usually not allowed |
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Name four eukaryotic translation initiation models.
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cap, ires, poly(A) and cap + poly(A)
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What does IRES stand for, give the three steps, and name the type of translation initiation it is.
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internal ribosome entry sites / picornovirus rapidly shut-down host protein synthesis, viral proteases cleave IF-4G (polioviruses), viral-induced dephosphorylation of 4E-BP (EMCV) / cap independent
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Name three prokaryotic elongation factors and their eukaryotic equivalents.
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- EF-TU: EF1a
- EF-T's: EFIbetay - EF-G: EF2 |
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Name three prokaryotic protein translation termination factors and two eukaryotic ones.
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Prokaryotic organisms
1. RF1 (UAA, UAG) 2. RF2 ((UAA, UGA) 3. RF3 (GTP-binding) 1. RF1 2. RF3 (GTP binding) |
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Name seven eukaryotic translation control mechanisms.
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-Secondary structure of mRNA
-multiple AUG codons in 5’ UTR -micro ORFs in the 5’UTR -protein binding sites in 5’UTR and 3’UTR -*phosphorylation of eIF-4E and eIF-4EBP -*phosphorylation of eIF2 alpha subunit -*viral proteases * = global inhibition |
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Name four 5' UTR regulated transcription genes.
Name the type of transcription gene that is regulated by 3' UTR. |
ferritin, mammalian sperm protamine, Drosophila sperm genes, riboswitches
Large numbers of Drosophila development genes |
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What regulates the ternary complex?
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phosphorylation of eIF2 (phosphorylation of the SER51 on eIF2)
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Name three factor of the regulation of ternary complexes.
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-phosphorylated eIF2 binds 100x more tightly to eIF2b
-prevents GDP to GTP exchange -blocks attachment of Met-tRNAi to 40S ribosome |
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What does phosphorylation of eIF-2 do?
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makes genes inactive (turns off translation)
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How do we distinguish between the multiple eukaryotic eIF-2 kinases? Name four and what they respond to.
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All Phosphorylate Ser51 but in Response to Different Signals /
HRI: Heme deprivation / PERK: ER stress caused by accumulation of unfolded proteins / PKR: viral infection by double-stranded RNAs / GCN2: amino acid or glucose deprivation |
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Define the action of eukaryotic translation initiation.
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Involves Many eIfs, Several of Which (1A, 3 &2-GTP) Pre-bind the 40S Ribosome Subunit Together With the Initiator Met-tRNA to = 43S Complex that is Recruited to the mRNA thru the interactions of several other eIFs Already Bound to the mRNA.
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Why does eIF4E bind to 5' cap? Why does eIF4G link the cap to the poly A tail?
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to remove dsRNA / to effectively give rise to a Circular mRNA
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What happens after 43S complex is bound?
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it “scans” along mRNA until it recognizes a sequence containing initiator AUG
(typically 5’-CCACCAUGC-3’ KOZAK Sequence) / Then eIF2-GTP->GDP, eIFs are released to allow entry and binding of 60S Ribosomal Subunit |
