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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
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
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
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
- 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
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
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
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
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
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
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
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
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
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
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
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
Why do Rfs and Efs use the same ribosomal site for their actions?
Molecular mimicry - all the factors have the same general shape
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
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
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
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