MCB 250 Fall 2022 Discussion 9 Oct 27 Nov 1 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 Lac It Lacpt Loco Lac operon always activated Laci cant bind to taco Laci B A mutation in the CAP binding site DNA prevents it from binding CAP protein CAP is catabolite activator protein C A mutation in the lacI gene prevents the LacI protein from binding to allolactose Allolcctose binds to Laci it to inhibit B galactosidase synthesis preventing 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 transcription state increases Sigma to recruits the RNA polymerase 4 2 Regarding bacterial gene regulation A What is the definition of basal transcription in E coli and other bacteria basal transcription is normal transcription level or 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 strong high level Of RNA polymerase binding to the promoter region weak low affinity to RNA polymerase 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 low affinity weak promoter low rate OF transcription After experiment if there is higher number of transcripts that is visible then it is strong if not then its weak cid 0 3 In E coli some core promoters have either a CAP binding site or an UP Element in the DNA sequence immediately upstream of 35 An UP Element is a DNA sequence that provides an additional binding site for RNA polymerase and is generally associated with strong promoters Both DNA sequences can increase the rate of transcriptional initiation from the associated promoter increases RNA transcvistwr binding OF ENA polymerase A What is similar about the way in which these two DNA sequences increase the rate of transcription both up elements and cap binding site are rich in Att sequences which enhances holoenzyme to the promoter site B What is different about the way in which these two DNA sequences increase the rate of transcription VP elements directly binds to alpha binds with CTD White CAP binding cyclic AMP this by allowing binding Of trcnsc Factors 4 You encounter a strain of E coli that is unable to grow or divide using lactose as a carbon source You analyze the chromosomal genotype of these bacteria and determine it to be lacI lacP lacO lacZ lacY lacA You want to introduce an F plasmid into this bacterium to restore normal lac operon function i e all three protein coding genes will be repressed in the absence of lactose and be induced in the presence of lactose What is the minimum set of genetic elements that you must include on your plasmid in order to achieve this result tact codes inhibitor regulatory genes structural genes of IGC Iacp codes for promoter Iac operon operator For for of ICCO codes ACZ tact tact operon the minimum set OF genetic elements that must be included on plasma are 104 ICCZ