Please help answer. all the questions 21. Several experiments were conducted to
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Question
Please help answer. all the questions
21. Several experiments were conducted to obtain information about how the eukaryotic ribosome recognizes the AUG start codon. In one experiment, the gene that encodes methionine initiator tRNA (tRNAi Met) was located and changed. The nucleotides that specify the anticodon on tRNAi Met were mutated so that the anticodon in the tRNA was 5’–CCA-3’ instead of 5’-CAU-3’. When this mutated gene was placed in a eukaryotic cell, protein synthesis took place, but the proteins produced were abnormal. Some of the proteins produced contained extra amino acids, and others contained fewer amino acids than normal. What do these results indicate about how the ribosome recognizes the starting point for translation in eukaryotic cells? Explain your reasoning, and also why some proteins were missing amino acids, and some had extra amino acids.
22. Compare the observed result for the eukaryotic experiment in problem #21 to what you expect the outcome would be if the experiment were performed in bacteria. Would the result be the same or different? Explain.
23. Assume that a particular miRNA (mir-77) regulates a single target gene, lag- 3. Assume that the normal function of lag-3 is to promote cell division. When it is active, the cell will initiate DNA replication (proceed through the G1 checkpoint).
a. What is the expected phenotype for lag-3 (null) mutants?
b. What is the expected phenotype for mir-77 (null) mutants?
c. What would be the expected double (null) mutant phenotype? Would it look like the mir-77 mutant or the lag-3 mutant?
24. There are many large non-coding RNAs expressed from the genome (linc genes). They are classified as non-coding because they lack an obvious open reading frame, or they have only a very small open reading frame (relative to the length of the RNA). These RNAs include many genes that function as RNA, such as Terc (an RNA in the telomerase) and RNAse P (a ribozyme). However, the function of many of them is unknown. A recent study of ribosome occupancy reported that there are ribosomes associated with these RNAs, suggesting that they may encode functionally relevant proteins or peptides. Assume you are studying one of these linc genes, and when the gene is deleted from the genome you observe that the cells are auxotrophic for uracil (they will not grow in the absence of uracil). Your goal is to investigate whether the endpoint assay for gene activity (to promote uracil production) is mediated by the RNA itself, or through a protein product translated from the RNA. What sort of experiment might you do to test whether the small open reading frame in the linc gene produces a protein product that is important for synthesis of uracil?
25. The following figure represents data from an analysis of sequence present in Ribosome Protected Fragments. Ribosome/RNA complexes are treated with RNase I, which preferentially degrades away the unprotected RNA (RNA not covered by a ribosome). This RNA is then subjected to RNAseq analysis.
a. The “RNA input” is also illustrated in the figure (it is indicated as “-“ to
generate the image, but the values are positive, like they are for the experiment),
and it serves as a control. Why might this be an important control?
b. There are two “peaks” in the RPF data that are highlighted, one upstream and
one downstream of the coding sequence (CDS). Explain the significance of
these peaks. What do they tell you about the rate of ribosome movement across
the RNA?
Explanation / Answer
21. The ribosome recognises the start codon near the 5' end of the mRNA where there are specific translation sites present that incorporate the mRNA onto that site . These sites are known as translation initiation sites. The 5' terminal sequence has an untranslated region that consists of the non coding sequence. Following this is the start codon , which is AUG Coding for methionine. The factors eIF-1, eIF-1A, and eIF-3 bind to the 40S ribosomal subunit, and the factor eIF-2 gets associated with the initiator methionyl tRNA. Also, eIF-4G, binds to eIF-4E and to poly-A binding protein or PABP, which is associated to the poly-A tail at the 3’ end of the mRNA. Thus, the initiation factors in eukaryotes recognise both the 5’ and 3’ ends of mRNAs.
When , the initiation codon is not recognised correctly, there is a total distortion of the translation machinery, which also recognises the 3G' end, thus leading to incorrect polypeptide sizes, either smaller or larger than usual.
22. The result in prokaryotic experiment would be different. The prokaryotes have many prokaryotic mRNAs that would encode for multiple polypeptides that are synthesized independently from distinct initiation sites. Such an mRNA is called a polycistronic mRNA, which is present in prokaryotes , but not in eukaryotes. In prokaryotic and eukaryotic cells, the signals that identify initiation codons are different. This is also because of the distinct functions of both polycistronic and monocistronic mRNA. Initiation codons in bacterial mRNAs have a preceding Shine-Delgarno sequence , that correctly aligns bacterial mRNAs on ribosomes.
23.
a. What is the expected phenotype for lag-3 (null) mutants?
Ans: Phenotype for lag-3 (null) mutants, would be restricted .
b. What is the expected phenotype for mir-77 (null) mutants?
Expected phenotype of null mir 77 cells would also be similar to those of null lag 3 mutants.
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