Question

PROBLEM SET #3 MAKEUP DUE: If a gene is cloned and then expressed in vitro (via a coupled transcription/translation system) the expectation is that a single polypeptide will be observed when analyzed by SDS Page (e.g., at 60KD in lane 1). Furthermore, if a premature stop codon were engineered 83% of the way down the gene, then protein synthesis would stop prematurely and a single polypeptide would be expected at 50KD (lane 2). 60kD 50kD 40kD 30kD In a number of cases, a "gene" was thought to be cloned, but when that "gene" was expressed in vitro in fact two proteins were observed via SDS Page A region from Bacillus subtilis was cloned and found to produce a 60KD protein and a 40KD protein as shown in the left panel. The proteins were assayed and two activities (TilS and HprT) were found. TilS does a covalent modification on tRNAe, while HprT is involved in a purine salvage pathway. The activities of TilS and HprT are given under the left panel. A premature stop codon was placed in one position in the clone to give the results in Lane 2, while a second premature stop codon was placed in another place and gave the results in Lane 3. Notice how the activities change Draw how the gene(s) must be structured. What is the MW of the TilS protein? What is the MW of the HprT protein? The analogous region from the related organism Listeria monocytogenes was also cloned and gave the results shown in the right panel. The same two activities (TilS and HprT) were assayed and gave the results shown under the right panel A premature stop codon was placed in one position in the clone to give the results in Lane 2, while a second premature stop codon was placed in another place and gave the results in Lane 3. Notice how the activities change. В. subtilis L. monocytogenes 100kD 100kD_ 80kD 80kD 60kD 40kD 20kD 40kD_ 20kD 0kD 0kD TİÏS HprT + Tils HprT + How can the results in the right panel be rationalized? Assume that MW values observed in gels are only approximate Imagine that this region of the Listeria monocytogenes genome evolved from the same region in Bacillus subtilis Propose what kind of mutation could have given rise to the change in behavior Transcription Signals (above the line) Translation Signals (below the line)

Answer 1