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129 Cards in this Set

  • Front
  • Back
Prokaryotes translation info.
1. Single form of RNA polymerase (5-6 subunit enzyme that does all the transcription for the cell)
2.Introns are rare (do not have codons)
3. polycistronic messages
4. Little RNA processing
5. few transcription initiation factor
eukaryotes translation info.
1. multiple forms of RNA polymerase (RNAPol I, II, III)
2. introns common
3. monocistronic (one message, one mRNA, one protein..)
4. Extensive RNA processing
5.multiple transcription initiation factors
sigma subunit (recognizes -10,-35 region) combined with core enzyme (alpha, beta and beta prime subunits)
rho independent transcriptional termination
unstable complex formation
-RNA polymerase slows down
-G-C rich regions: tighter interaction, slows it down
-hairpin region of RNA
rho dependent
does not happen at termination sequences. recognizes certain sequences and moves in ATP-dependent manner and moves up RNA to disrupt transcription
-also occurs in high G-C areas
agents that bind to RNA polymerase and inhibit transcription
1. Rifampin-binds to beta subunits in prokaryotes
3.alpha amanitin (only euk binds to Pol II)
4. heparin (stops transcription in test tube)
agents that bind to DNA template
actinomycin D
ways to inhibit transcription
1. bind to RNA polymerase
2. bind to DNA template and get in between interaction
found in prokaryotes: transcription unit in which several genes are cotranscribed
DNA binding proteins
bind to promoters in euk and prok and direct RNA polymerase to promoter site
explain euk transcription factors
1. binding of RNA Pol II requires basal trancription factors-TFII
2.TBP (TATA binding protein:subunit of TFIID) binds to TATA box
3. TFIIB and TFIIA interact with TBP
4. RNA Pol II binds the complex and DNA aligned at startpoint.
5. TFIIE,F and H bind and ATP is cleaved and transcription is initatied
increase rate of transcription
-typically bind upstream. bind to transcriptional activators
transcriptional activators that interact with basal transcriptional complex, forming loops in DNA.
-become active when something needs to be transcribed
may interact with transactivators to allow to bind
capping of mRNA
-occurs cotranscriptionally
-can be produced from GTP
-binding of mature RNA to ribosome during protein synthesis
poly-a tail
-attached at 3' end
-primary transcript is cleaved 10-20 nucleotides downstream
-no poly T sequence in DNA template
-polyA added posttranscriptionally
cleavage and polyadenylation specificity factor
-binds polyadenylation sequence AAUAAA
-contains and endonuclease which catalyzes cleavage downstream of polyadenylation sequence forming new 3' end
-poly A polymerase can then bind
all start with 5' GU and end with 3' AG-not every one results in a splice site
deal with splicing of introns
-small nuclear ribonuclearproteins (snRNS's)
-rich in uracil
mature mRNA: where comes from, where goes
once it has its cap and tail, leaves nucleus to cytoplasm to meet with ribosomes to form proteins
ribosomal RNA processing
methylation and cleavage by endonucleases
-most are made in nucleolus by PolI
Euk tRNA
made by RNA Pol III
-cleaved at both ends by endonucleases
processing of euk tRNA
all receive 3'cap
-introns removed from anticodon arm by endonuclease and free ends ligated by ligase
-hydroxyl on 3' terminal adenylate residue forms ester with cognate aa
-charged tRNA now carries aa acid to ribosome for synthesis
3 common modifications of nucleotides in tRNA post transcriptionally
1. Uracil methylated by SAM to make thymine
2. dbl bond in uracil reduced to form dihydrouracil
3. N-glycosidic linkage of Uracil with ribose is changed to C-C linkage resulting in pseudouridine
alpha amanitin
-lethal toxin
-inhibits mRNA synthesis
-cyclic peptide structure
biochemical basis of beta-thalessemias
hereditary anemias
-reduced production of hemoglobin
-most common genetic disorder around the world
-affect synthesis of beta globin chains
-mutations in promoter
-mutations in the cleavage and polyadenylation
works in wobble hypothesis in translation. can bind with U,C or A on mRNA. Inosine is on 5' position of anticodon
-allows for less than 61 tRNA's
silent mutation
when aa sequence not affected
missense mutation
aa is changed
nonsense mutation
causes early termination
one or more nucleotides are added to DNA (jumping gene, translocation)
frameshift mutation
occurs when insertion or deletion is not a multiple of three nucleotides. Usually causes early termination.
-used as defense sometimes because draws attention to where DNA needs to be fixed
aminoacyl tRNA synthetase
covalently attached aminoacyl tRNA to 3' end of each tRNA. Different one for each aa. Charges (aa attaches) the molecule and ensures fidelity
initiation factors
required to form a complex; recognizes start codon and positions all players.
-prok (IFI, II, III)
-euk (EIF's, twelve or more)
4 steps of euk translation initiation
-eIF-4 (cap binding protein) CBP binds to 5'cap of euk mRNA
-initiation complex (40S ribosome, aminacylated initiator tRNA and other EIF's searches mRNA from 5'-3' looking for initiator codon
-Met-tRNAiMet complexed with EIF2 and GTP binds to AUG
-GTP hydrolyzed, EIF's released and 60S ribosome binds to complete the ribosome
-during initiation Met-tRNAiMet iis bound to ribosomal P site
translation initiation in prok
-prok mRNA not capped!
-30s ribosomal unit binds to Shine-Delgarno sequence on mRNA upstream of initiation sequence. Start codon will be nearby downstream
-Shine-Delgarno sequence binds to complementary base sequence at 3' end of 16S rRNA in 30S ribosomal subunit.
three steps of elongation
1. positioning of correct aa-tRNA in ribosomal A site
2. formation of peptide bond, catalyzed by peptidyl transferase
3. translocation or movement of mRNA by one codon, such that tRNA carrying peptide chain moves from A-site to P-site
first step necessary in elongation
1. elongation factor EF-1 in euk, EF-Tu in prok and hydrolysis of GTP are required
second step in elongation
formation of peptide bond is catlyzed by peptidyl transferase contained within the large ribosomal subunit
-RNA catalyzed reaction: ribozyme
-bond formed between aminyl group of aa and carbonyl group at P site
third step in elongation
-new peptidyl tRNA group moved from a to P site
-the deaminoacylated tRNA has shifted from P to E site
-EF2 in Euk or EFG in prok and GTP are required.
termination of tranlation
release factor binds instead when stop codon encountered
sickle-cell anemia
missense mutation
-GTG replaces GAG
-causes Val to replace Glu
-hydrophobic interactions b/w val cause precipitation
regulation of globin by heme
heme keeps initiation factor eIF2 in the active state so that initation and synthesis of globin can proceed
diphtheria toxin
B subunit binds at the cell membrane facilitating entry of A subunit
-inhibits translocation
-leads to inhibition of protein and eventually cell death
peptide held in place by many weak interactions with ribosome, but will start folding as they come out. some proteins need aid of chaperones to fold correctly.
how do chaperone work
bind hydrophobic regions of nacent protein to avoid aggregation
-maintain unfolded state to allow passage through membranes
-prevent incorrect folding intermediates
-prevent inappropriate interactions with other cellular proteins
9 common posttranslational modifications
1. deformylation of N-terminal residue in pro
2.removal of N-terminal Met
3.formation of disulfide bonds
4.cleavage and activation by proteanases
5. acetylation of amino terminus
6. methylation of lysine
7. hydroxylation of lysine and proline
8. phosphorylation of serine, threonine, tyrosine
9. O-linked and N-linked phosphorylation
exported proteins
proteins destined for export are synthesized on membrane-bound ribosomes of the rough ER
mitochondrial proteins
majority synthesized on cytosolic ribosomes and imported in to the mitochondria
-have n-terminal sequences that facilitate transport in to the mitochondria
membrane bound ribosome
some proteins enter rough ER as they are being synthesized
-N-terminal sequences containing hydrophobic aa called the signal peptide
-signal recognition particle (SRP) binds the signal peptide and ribosome, inhibiting translation
- SRP directs ribosome to rough ER by binding the SRP receptor or docking protein
-translation continues and polypeptide enters lumen of RER
-signal peptidase cleaves the signal peptide.
processing of proteins synthesized and translocated in the ER
-transferred to Golgi complex in vesicles
-glycosylation of the protein may occur in Golgi or RER
proteins made in RER are destined for where
1. lysosomes
2.cell membrane
3. secretion
signal telling transport vesicle to go to lysosome from Golgi
I-cell disease
mannose-6-P can't be attached to hydrolytic enzme
-lysosomes swell
-see lysosomes in blood, where normally don't because the cells burst
-usually death by age 8
4 inhibitors of protein synthesis
1. streptomycin (initation, elongation)
2. tetracyclins (aminoacyl tRNA binding to A site)
3. chloramphenicol (peptidyltransferase)
4. erythromycin (translocation)
two ways transcriptional regulation implemented
1. through molecules that bind RNA polymerase
2. through molecules that bind the DNA template
bind at operators and inhibit ability of RNA Pol to do its job and transcribe the operon
binds to repressor so that it can bind to allow inhibition of transcription
ex: Arg. presence goes up like all other proteins, but if Arg is added (corepressor), synthesis will cease
observation of sugar utilization enzymes
cells growing and lactose is added. at this point enzymes to chew up lactose begin to be made: due to repressor sitting blocking transcription and inducer (lactose) cause transcription to continue
affects of AA and sugars on transcription
-AA present turns it off
-Sugar present turns it on
regulation of genes at transcription level
-where most regulation occurs
-transcription factors either activate or repress promoter activity
regulation at level of DNA
-gene loss
-gene amplification
-DNA rearrangements to produce different proteins
-base modification: methylation of DNA
-chromatin condensation: no transcription occurs in dense (heterochromatin) regions
gene regulation at level of transcription
-inducible gene expression
-assembly of basal transcription complex
-TBP (TATA Binding Protein) recruits other necessary factors
-general transcription factors: part of the basal transcription complex
-specific transcription factors: gene specific
specific transcription factors
ex: thyroid and steroid hormone receptors
-promote assembly of basal transcription process either alone or with coactivators and thereby increase the rate of transcription
transcription activation by glucocorticoid vs. thyroid hormones
thyroid hormone interacts with the repressor once inside the nucleus
DNA binding domains
zinc finger
leucine zipper
4 modes of actions of transcriptional repressors
1. activation
2. repression by competition
3. repression by quenching
4. active repression
what effects the stability of mRNA
3' polyA tail
5' cap
sequences in 3' end untranslated region of some mRNA
transferrin receptor
-with adequate cellular iron, transferrin receptor rapidly degraded b/c iron inactivates IRE-BP
what is ferrritin (ex of regulation at translation)
iron storage protein, required when iron levels are high. has hairpin loops that bind regulatory proteins and prevent initiation of translation
-high iron: binds regulatory proteins and reduces affinity for 5' IRE-BP and ferritin synthesis proceeds
-low iron levels: regulatory proteins bind 5' IRE and blocks ferritin synthesis
correlation b/w globin protein and heme
-heme high: protein is formed through EIF2 actiatioin
-heme low: kinase is activates which deactivates EIF2 and protein synthesis
restriction endonucleases
Endonucleases—cut double stranded DNA at specific sequences. Used in cloning and some diagnostic assays (e.g., RFLP). Recognition site usually about 6bp. Can cut out what’s important and move to a different place
DNA polymerase
polymerizes a new DNA molecule that is complementary to an existing template strand. It has many uses in molecular biology.
DNA ligase
forms a phosphodiester linkage between the 5’-phosphate and the 3’-hydroxyl of adjacent nucleosides on “nicked” DNA. Used in cloning.
RNA polymerase
polymerizes an RNA molecule that is complementary to an existing DNA template.
where does glycosylation of protein occur?
lumen of RER or golgi
function of mannose-6 phosphate
signal telling transport vesicle to go to lysosome
I-cell disease
Defect in lysosomal enzyme targeting due to a deficiency in a glycosyltransferase enzyme. Therefore, the mannose-6-P cannot be attached to the hydrolytic enzymes.
-The lysosomes fill up with material that cannot be digested properly…lysosomes swell. Ends in early death
responses to glucose
glucose levels high, cAMP low
-CAP acts globally to enable transcription at promoters of several operons when glucose levels are low
miniature gene. Gets transcribed first: leader sequence upstream
-Trp required to make leader sequence in Trp operon
Hairpin on RNA in regards to Trp
-4 regions that can form hairpin structure on mRNA.
-region 3 to 4: looks like terminator
-if no Trp, regions 2 and 3 interact and without terminator, RNA Pol continues through transcription
principle point where gene expression is regulated
initation of transcription
gene regulation at level of DNA
-base modification: methylation
-chromatin condensation
gene regulation at level of tanscription
-inducible gene expression: inducers bind to receptors which bind to specific response elements
-assembly of basal transcription complex
-general transcription factors
-specific transcription factors (transactivators)
difference in glucocorticoid and thyroid hormone in terms of interaction with repressor
thyroid hormone interacts once inside nucleus whereas the glucocorticoid interacts with a hormone receptor which then uses a nuclear localization signal
specific transcription factors
-(eg. thyroid hormone receptor, steroid hormone receptors) promote the assembly of the basal transcription complex either alone or with coactivators and thereby increase the rate of transcription
-have DNA binding domain and transactivating domain
4 DNA binding domains
1. zinc finger
2. helix-turn helix
3. leucine zipper
4. helix-loop-helix
4 modes of action by of transcriptional repressors
1. activation
2. repression by competition
3. repression by quenching
4. active repression
RNA editing
post transcriptional modification: similar to mutation, but in RNA. alteration of bases in RNA transcript
transferrin receptor
with adequate cellular iron: rapid degradation because iron inactivates IRE-BP
-with low cellular iron: active IRE-BP prevents degradation of tranferrin receptor mRNA by binding 3' end IRE
Recombinant DNA Biotechnologyy
Production of Therapeutic Proteins
Medical Diagnosis
Disease diagnosis
Predict risk of genetic disorders
Determine parentage and other relationships
Gene Therapy
molecular genetics technics
Cloning DNA (Recombinant DNA)
Polymerase Chain Reaction
Nucleic Acid Electrophoresis

DNA Hybridization
Transgenic animals
Restriction Fragment Length Polymorphism
restriction endonucleases
cut double stranded DNA at specific sequences. Used in cloning and some diagnostic assays (e.g., RFLP). Recognition site usually about 6bp. Can cut out what’s important and move to a different place
DNA polymerase
polymerizes a new DNA molecule that is complementary to an existing template strand. It has many uses in molecular biology.
DNA ligase
forms a phosphodiester linkage between the 5’-phosphate and the 3’-hydroxyl of adjacent nucleosides on “nicked” DNA. Used in cloning.
RNA polymerase
polymerizes an RNA molecule that is complementary to an existing DNA template.
reverse transcriptase
polymerizes a DNA molecule that is complementary to an RNA template. It is used in the production of cDNA and to identify transcriptional start-sites of genes.
uses of nucleic acid hybridization
Southern Blotting (detecting DNA)

Northern Blotting (detecting RNA)

Genetic Disease Diagnosis- using in quick reagent access in order to get results right in office.

Pathogen Identification

Microarrays (assaying gene expression)
sources of DNA for cloning
Restriction digests of DNA from various sources

cDNA libraries
Collection of genes produced by mRNA b/c all introns are already cut out and has polyA tail. Use reverse trancriptase to produe DNA. Use polyT as primer.
PCR products
PCR consists of:
DNA template

Oligonucleotide primers


Heat-stable DNA polymerase
Ferritin (Ex of regulation at level of translation)
is an iron storage protein, required when iron levels are high. Ferritin mRNA contains hairpin loops at the 5’-end that bind regulatory proteins (IRE-BP) preventing initiation of translation
High cellular iron levels -. Iron binds the regulatory proteins reducing their affinity for the 5’ IRE and ferritin synthesis proceeds
Low iron levels - the regulatory proteins bind the 5’-end IRE, blocking ferritin synthesis
Globin production (ex of regulation at level of translation)
Heme acts by preventing eIF2 phosphorylation
Binding of heme to eIF2 kinase, inactivates the kinase, keeping eIF2 active
ubiquitin pathway
pathway-proteins selected for degradation are bound covalently by a small protein ubiquitin, then degraded by a complex of three enzymes
cytoplasmic proteases
means of protein degradation: Ca dependent
the most common types of cancer, arise from the cells that cover external and internal body surfaces. Lung, breast, and colon are the most frequent cancers of this type in the United States.
supporting tissues of the body such as bone, cartilage, fat, connective tissue, and muscle.
lymph nodes and tissues of the body's immune system.
immature blood cells that grow in the bone marrow and tend to accumulate in large numbers in the bloodstream.
2 ways cancer spread through body
Invasion refers to the direct migration and penetration by cancer cells into neighboring tissues.

Metastasis refers to the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body.
benign tumors
Moles (nevi) are tumors of the skin

Mostly melanocytes with pigmentation (melanin).

UV light can convert benign moles to malignant melanoma (environmental factors discussed later
known causes of cancer
Some cancers (~ 10%) are strongly genetically predisposed by inherent germ line mutations behaving in Mendelian traits (heredity basis).

Most cancers are genetic diseases of somatic cells (normal body cells) that lose growth control. Spontaneous.

Studies indicate three main categories of factors that contribute to the development of cancer are:
Chemicals (smoking, toxins)
Radiation (sunlight)
Viruses or bacteria.
human cancer viruses
Among the retroviruses HIV does not cause cancer.
ebstein-barr:burkitt's lymphoma
HPV: cervical
hep B: livr
human T-cell lymphatrophic: adult T-cell leukemia
kaposi's sarcoma: kaposi's sarcoma
helicobactor pylori
can cause stomach ulcers
may be increaesd risk of stomach cancer
tumor viruses
Tumor viruses obtain their oncogenes from host cells they have infected.

Initially, viral genome is incorporated into host genome. When viral genes are expressed and new viruses are produced, some host genes are carried by the viral genome
Src is a proto-oncogene as well, but gets changed in some way to where it is no longer under control.
Oncogenes arise from proto-oncogenes that have been damaged.

Almost always, oncogenes are gain of function mutants: they either result in a protein with increased activity, or they increase the expression of normal proteins
Oncogenes are genes whose PRESENCE and / or over-activity can stimulate the development of cancer.
Oncogenes can be activated even if only one allele is affected.
tumor suppressor genes
normal genes that control cell growth. Their ABSENCE can lead to cancer. Both alleles must be affected to lose tumor suppressor gene function.
DNA repair enzymes
critical for repairing DNA damage before a cell divides. Unrepaired DNA damage will propagate through DNA replication.
Xeroderma pigmentosum
mutation in genes of the nucleotide excision repair pathway.
Hereditary nonpolyposis colorectal cancer (HNPCC)
mutation in mismatch repair genes: get mismatch repair, cells can’t fix it and it is inherited
Philadelphia chromosome
In bone marrow cells it is possible for DNA to exchange between chromosomes 9 and 22.

This exchange forms the “Philadelphia Chromosome.” A portion of ABL (9) inserts into BCR : Breakpoint Cluster Region(22)

The hybrid BCR-ABL gene produces a fusion protein that has increased tyrosine kinase activity and promotes uncontrolled growth of leukemic cells.

Cells also lose apoptotic control: BCR controls apoptosis.

Causes chronic myelogenous leukemia (CML). One cell is all it takes…
cellular distribution of oncogenes
if on cell surface: receptors (PDGF, RET, KIT, EGFR, CSFRI,
inside: transcription factors, or interact with them
Ras-activated MAP-kinase pathway
Ras is a G-protein that activates raf, raf is a kinase that activates MEK by phosphorylating it. MEK-P phosphorylates MAP-kinase. MAP-K-P has several targets.
Mutations in any of the proteins that regulate the MAP-kinase activity, or any of the proteins induced by MAP kinase activation, can lead to uncontrolled cell proliferation.
Ras is a G-protein (see slides 27 and 32 in lectures 19-20)
Binds GDP, is inactive and bound to a growth factor receptor.

Growth factor binding to the receptor activates ras, induces it to exchange GDP for GTP.

GTP-ras binds to raf protein (kinase) and activates it. In the process GTP-raf becomes GDP-raf (inactive)

Raf now phosphorylates downsteam targets  cell proliferation.

Known mutations in raf allow it to bind GTP at all times, thus stays active at all times, stimulating raf continuously.

Overactive ras involved in colon and bladder cancers
tumor suppressor
Affect cell cycle regulation, signal transduction, transcription and cell adhesion.

Products of tumor suppressor genes often modulate pathways that are activated by the products of proto-oncogenes.
p53 stops replication in cells that have DNA damage and targets the unrepaired cells for apoptosis.

DNA damage stimulates production of p53 protein.

P53 (finds areas of damage and binds: becomes a signal saying that it has found DNA that is damaged) stimulates production of p21, which inhibits cyclin/CDK complexes and stops cell cycle progression and cell proliferation.

p53 stimulates production of DNA repair enzyme GADD to fix the DNA problem.

If DNA is not repaired, p53 stimulates activates two genes for apoptosis: bax and IGF-BP3
tumor suppressor and transduction
GDP-ras is the inactive form that binds to the growth factor receptor and waits for a signal.

GTP-ras is activated form, binds to raf to stimulate cell proliferation.

Tumor suppressor gene protein neurofibromin (NF-1) binds to ras and holds it in the GDP-ras form.

If NF-1 is missing, GDP-ras will stay active longer than needed  cell proliferation.
three phases of apoptosis
initiation (above), signal integration (apoptosis signals balanced against anti-apoptotic mechanisms), and execution phase (cells degraded by caspase enzymes).
act to maintain the length or to lengthen telomeres
not active in normal somatic cells, but active in most tumor cell lines

Through normal replications, chromosomes in cells shorten to a critical length, then cell enters apoptosis.

In cancer cells, telomerase keeps chromosomes at full length, avoiding the apoptosis signal