RNA Polymerase and CI Regulator Interactions in Gene Regulation in Bacteriophage Lambda Re-visited

Authors

• Z Wang
• J Chen
• D Kiesewetter
• RD Leapman
• A Jin

Abstract

Background: The bacteriophage lambda paradigm in gene regulation revealed several basic principles of transcription regulation. After infection with E. coli, lambda follows either a lytic growth or a lysogenic growth cycle. The CI represses the lytic promoters (PR and PL) and activates and represses the lysogenic promoter (PRM) at low and high concentrations, respectively. CI regulates PRM by binding to tripartite, OR1, OR2 & OR3 located between PRM and PR. CI binds to OR1 and OR2 represses PR. CI bound to OR2 activates PRM through direct contact between RNAP~PRM and CI ~OR2. CI binding to OR3 represses PRM.
Methods: In an in vitro transcription system, we analyzed the RNA polymerase and CI interactions by monitoring PRM levels by using DNA and CI mutants.
Results: We obtained unexpected findings. First, DNA mutants resulted in PRM repression. This repression is depending on DNA looping and CI~OR2. CI mutant, E34K, resulted in PRM repression at the same CI concentration for PR repression.
Conclusion: Attempted disruption of the activation complex between RNAP at PRM and CI at OR2 by mutating CI or inserting or deleting base pair to change the angular orientation and distance between RNAP and CI unexpectedly led to CI-dependent repression of PRM. From these unexpected results, we propose that under all three conditions, the OR2 bound CI is creating an inhibitory contact with RNA polymerase at PRM preventing the latter to escape the promoter and repressing transcription. Studies are being conducted to test the model.

Scientific Focus Area: Molecular Biology and Biochemistry

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