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What are the sizes of the ribosomal subunits for prokaryotes and eutaryotes?
- Bacterial - 70S = 50S + 30S
- 1. 50S = 23S + 5S
- 2. 30S = 16S
- Mammalian - 80S = 60S + 40S
- 1. 60S = 28S + 5.8S + 5S
- 2. 40S = 18S
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What is the flow of tRNA through the ribosome?
- 1. Acelated t-RNA enters A-site
- 2. Peptidyl tRNA bound in P-site
- 3. Deacylated tRNA exits via E-site
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Summarize the error rates at the steps that can affect the accuracy of translation
- 1. Wrong base on mRNA - 10-6
- 2. Frameshift - 10-5
- 3. Wrong aminoacyl-tRNA - 10-4
- 4. Wrong amino acid - 10-5->-4
- 5. Wrong tRNA - 10-6
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What is the role of Shine-Dalgarno sequence, 30S subunit and ribosome cycle, and IF-1, -2, -3 in prokaryotic translation initiation?
- 1. The Shine-Dalgarno sequence is complementary to a conserved sequence of rRNA ~10 bp upstream of the start codon and tells ribosome which aug is for translation
- 2. Free 30S subunit binds to mRNA only when bound to IFs
- 3. IF-1 - binds near the A site and prevents aminoacyl-tRNA from entering
- 4. IF-2 brings tRNA to P site
- IF-3
- 1. The pool of 70S ribosomes is in equalibrium with free ribosomal subunits
- 2. 30S subunit bound with IF-3 can bind mRNA but not 50S subunit
- 3. IF-3 must be released before 50S subunit can join 30S
- 4. It checks the accuracy of recognition of the first aminoacyl-tRNA
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What are the key structural features of the initiator tRNA that insure it can only be used for initiation?
- 1. It has a formylated Met which is recognized by IF-2
- 2. The stem is unpaired which is required for formylation
- 2. 3 G-C pairs in the stem preceding the anticodon loop are required for it to be inserted in the P site
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Describe the role of the 40S ribosomal subunit in eukaryotic translation initiation
- 1. 40S binds to methylated 5' cap
- 2. 40S migrates to initiation site that includes AUG
- 3. IRES (internal ribosome entry site) - in certain viral RNAs, it bypasses all AUGs in the 5' UTR
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What are the major functions of the major eIFs (eIF1-5)?
- 1. eIF2 binds Met-tRNA to 40S
- 2. eIF3 and eIF1 keep free 40S subunits dissociated
- 3. eIF4 binds the mRNA 5' cap and polyA tail
- 4. eIF5 helps large subunit bind small subunit
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Describe the function of EF-Tu and its cycling on and off the ribosome.
- 1. EF-Tu-GTP brings aminoacyl-tRNA to the A site on 30S
- 2. GTP is hydrolyzed to GDP in the process of binding the CCA end to the 50S A site
- 3. Ef-Tu and GDP are released and recycled
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What is the action of GMP-PCP and kirromycin at the elongation step?
- 1. GMP-PCP - an analog GTP that binds aa-tRNA and allows it to bind to the ribosome but not to the A site (GTP is required)
- 2. Kirromycin - an antibiotic that prevents EF-Tu-GDP form being released from the ribosome
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What is the peptidyl transferase rxn and where is it located?
Peptide bond formation takes place by rxn between the polypeptide of peptidyl-tRNA in the P site and the amino acid of the aa-tRNA in the A site
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Why does the antibiotic puromycin function as a chain terminator of translation?
- 1. Puromycin mimics aa-tRNA because it resembles an aromatic aa linked to a sugar-base moiety
- 2. Puromycin isn't anchored to the A site of the ribosome and thus released prematurely, terminating translation
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What is translocation?
- 1. The cycle of addition of aa to the growing polypeptide chain via the ribosome advancing 3 nucleotides along the mRNA
- 2. The peptidyl-tRNA is in the P site; the aa-tRNA is in the A site; the de-aa-tRNA is in the E site
- 3. The 50S moves relative to the 30S followed by the 30S moving relative to the mRNA
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What is the role of EF-G (translocase) in translocation, the action of the antibiotic fusidic acid, and GMP-PCP at this step in translation
- 1. Translocase catalyzes translocation of t-RNA at the end of elongation
- 2. Fusidic acid stabilizes translocase on the ribosome preventing it from being released and the next aa-tRNA can't bind
- 3. Translocase can still bind the ribosome when GMP-PCP is substituted for GTP
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What are release factors, what do they recognize, and where and when do they perform their function?
- 1. They recognize termination codons
- 2. RF1 recognizes UAA and UAG; RF2 recognizes UGA and UAA
- 3. They act at the ribosomal A site and require polypeptidyl-tRNA in the P site
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What is the function of RF3 and when is it needed in termination of translation?
It's role is to release RF1 and RF2 from the ribosome
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Why do Rfs and Efs use the same ribosomal site for their actions?
Molecular mimicry - all the factors have the same general shape
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Compare the formation of a peptide bond during elongation to its hydrolysis at termination.
Both transfer an OH from H2O, hydrolyzing the peptide-tRNA bond
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What are the remaining termination events once the polypeptide chain has been hydrolyzed from peptidyl-tRNA?
- 1. RRF (ribosome recycling factor) releases the last tRNA
- 2. EF-G releases RRF
- 3. Ribosome dissociates
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How can aa-tRNA bind adjacent to peptidyl tRNA in the ribosome?
mRNA turns between the P and A sites allowing aa-tRNA to bind adjacent codons
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What are the relative positions and names of the active sites in the ribosome?
- 1. A site and P site are angles with regard to one another
- 2. E site lies beyond the P site
- 3. Peptidyl transferase site stretches across teh tops of the A and P sites
- 4. Ef-Tu/G binding sites are at the base of the A and P sites
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