MCB250_Discussion 9_ WS9_FA23 - Taigue Woods

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Dec 6, 2023

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MCB 250 Fall 2023 Discussion 9 October 26-31 1. In wild-type E. coli , the lac operon can shift back and forth between three different states of transcription - i.e. basal, repressed, or activated - in response to the chemical composition of its environment. What effect would each of the genetic changes listed below have on these three transcriptional states? A. A mutation in the lacO operator DNA prevents it from binding LacI protein. A mutation in the lacO operator that prevents LacI binding eliminates the repressed state of the lac operon, causing it to be constantly "on." However, in the absence of glucose, transcription can still be further enhanced by the cAMP-CAP complex. B. A mutation in the CAP binding site DNA prevents it from binding CAP protein. A mutation in the CAP binding site that prevents CAP protein binding would reduce the operon's activation in the absence of glucose. As a result, even when lactose is present and glucose is absent, the lac operon would not achieve its maximum transcriptional activation due to the lack of the cAMP-CAP enhancing effect. C. A mutation in the lacI gene prevents the LacI protein from binding to allolactose. A mutation in the lacI gene that prevents the LacI protein from binding to allolactose means the LacI repressor will always be active, regardless of lactose's presence. As a result, the lac operon would remain repressed even in the presence of lactose, preventing the genes from being transcribed even when lactose is available as a substrate. D. The wild-type lac operon core promoter matches the sigma 70 consensus sequence at 9 out of 12 base pairs (in the -10 and -35 sequences). Envision a mutation that causes the lac promoter to match consensus at 10 out of 12 base pairs. What effect

would such a mutation have on the rate of initiation of lac operon transcription in each of the three states? A mutation that causes the lac promoter to better match the sigma 70 consensus sequence would increase the rate of transcription initiation in all three states of the lac operon. The most pronounced effect would be seen in the activated state, with lesser increases in the basal and repressed states. 2. Regarding bacterial gene regulation: A. What is the definition of 'basal transcription' in E. coli and other bacteria? Basal transcription in E. coli and other bacteria refers to the low, default level of gene expression that occurs in the absence of specific activating or repressing regulatory influences. It represents the minimal transcriptional activity driven by the core promoter elements without enhancement or inhibition by regulatory proteins. B. In E. coli, some promoters are said to be 'strong' ( e.g. the rRNA operon promoter) whereas other promoters are said to be 'weak' ( e.g. the lac operon). What does this distinction mean in terms of the rate of initiation of basal transcription? In E. coli, "strong" promoters have a higher affinity for RNA polymerase and initiate transcription more frequently, leading to high rates of basal transcription. Conversely, "weak" promoters initiate transcription less frequently, resulting in lower basal transcription rates.

C. Consider an E. coli gene whose promoter contains -10 and -35 sequences and is transcribed by the housekeeping RNA polymerase, i.e. the core polymerase associated with the sigma 70 subunit. Given the DNA sequence of this promoter, how would you predict whether it is a strong or a weak promoter? Explain your reasoning. To predict the strength of an E. coli promoter based on its -10 and -35 sequences, compare these sequences to the sigma 70 consensus sequences (TATAAT for -10 and TTGACA for -35). Promoters closely matching these consensus sequences are likely strong, while those with significant deviations are likely weak.

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