Differential binding mechanisms of anti-sigma factors E. coli Rsd and bacteriophage T4 AsiA to E. coli RNA polymerase lead to diverse physiological consequences

AstraZeneca R & D, Bellary road, Hebbal, Bangalore, India; Molecular Biophysics unit, Indian Institute of Science, Bangalore, India

^* To whom correspondence should be addressed. Email: umender.sharma{at}astrazeneca.com.

	Abstract

Anti-sigma factors E. coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, ⁷⁰, of E. coli. Though, both of them are known to interact with the C-terminal region of ⁷⁰, the physiological consequences of this interaction are very different. This study was undertaken with the purpose of deciphering the mechanism by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to inhibition of E. coli cell growth to different extents. It was found that AsiA is more potent inhibitor of in-vivo transcription and thus causes higher inhibition of E.coli cell growth. Measurement of affinity constants by surface plasmon resonance (SPR) showed that Rsd and AsiA bind to ⁷⁰ with similar affinity. Data obtained from in-vivo and in-vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. Binding patterns of AsiA and Rsd with ⁷⁰ studied by Yeast Two Hybrid system revealed that region 4 of ⁷⁰ is involved in binding to both these anti-sigma factors; however, Rsd interacts with other regions of ⁷⁰ as well. Taken together, these results suggest that higher inhibition of E. coli growth by AsiA expression is probably, due to the ability of the AsiA protein to trap the holo RNAP rather than higher binding affinity to ⁷⁰.