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Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

AstraZeneca Research and Development, Bellary Road, Hebbal, Bangalore, India,¹ Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India²

Received 13 November 2007/ Accepted 12 March 2008

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, ⁷⁰, of E. coli. Though both factors are known to interact with the C-terminal region of ⁷⁰, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments 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. The binding patterns of AsiA and Rsd with ⁷⁰ studied by using the yeast two-hybrid system revealed that region 4 of ⁷⁰ is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of ⁷⁰ as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to ⁷⁰.

Published ahead of print on 21 March 2008.