PS_6_key_05 - Bio 115, Winter 2005 Problem set #6 February...

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Bio 115, Winter 2005 February 18, 2005 Problem set #6 1. You have prepared an antibody that recognizes the 5' cap of human mRNAs with a high affinity. You prepare a cell lysate and perform an immunoprecipitation. What sorts of molecules are likely to be present in the immunoprecipitate? Answer : Remember that mRNA is associated with many proteins. So, we should see, mRNA hnRNP proteins RNA polymerase (remember, capping is a cotranscriptional event) poly-A-binding proteins snRNPs ribosomes translation factors 2. How is the 5' cap important for mRNA stability? For translation? Answer : It protects mRNAs from degradation, and is bound by translation initiation factor eIF4f. 3. Where in the cell does capping of mRNAs occur? Answer In the nucleus, very early in the process of transcription. 4. What protein(s) binds the 3' end of mRNAs? Answer Poly-A binding proteins 5. Besides alternative splicing, how can a mRNA be used to generate multiple different proteins? Answer By 'recoding', use of selenocysteine tRNAs and ribosomal frameshifting. 6. What are some of the major differences between translation in prokaryotes and eukaryotes? Answer Eukaryotes identify the site of initiation via scanning. Eukaryotic RNAs are usually monocistronic. Eukaryotes use a cap dependent mechanism of translation. (see pg 226 of the reader for other differences) 7. What are some of the energy dependent steps in translation? Answer Charging of tRNAs, delivery of amino-acyl tRNAs to the A-site(EF-TU), translocation (EF-G), initiation (eIF4A helicase, eIF2, eIF3, scanning). - 1 -
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Bio 115, Winter 2005 February 18, 2005 Problem set #6 8. How do eukaryotes determine where to initiate translation? How can this be proved experimentally? Answer They scan to the first AUG, which is recognized by base-pairing between the anti-codon of the initiator tRNA and the AUG. This can be demonstrated by mutations in the AUG that abolish translation and by compensatory mutations in the anti-codon that restore translation at the now non-AUG site. 9. How does the peptidyl transferase reaction of translation procede? In particular, a) does protein synthesis procede from the amino-terminus or carboxy-terminus of the protein? amino-terminus to carboxy-terminus b) does the tRNA in the A site receive the nascent peptide, or does it donate it’s amino-acid to the nascent peptide? Answer
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PS_6_key_05 - Bio 115, Winter 2005 Problem set #6 February...

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