Influence of Flexible ``omega'' on the Activity of E-coli RNA Polymerase: A Thermodynamic Analysis

摘要

The Escherichia coli RNA polymerase (RNAP) is a multisubunit protein complex containing the smallest subunit, omega. Despite the evolutionary conservation of omega and its role in assembly of RNAP, E. coli mutants lacking rpoZ (codes for omega) are viable due to the association of RNAP with the global chaperone protein GroEL. With an aim to get better insight into the structure and functional role of , we isolated a dominant negative mutant of omega (omega(6)), which is predominantly a-helical, in contrast to largely unstructured native omega, and then studied its assembly with reconstituted core1 (alpha(2)beta beta') by a biophysical approach. The mutant showed higher binding affinity compared to native omega. We observed that the interaction between core1 and omega(6) is driven by highly negative enthalpy and a small but unfavorable negative entropy term. Extensive structural alteration in omega(6) makes it more rigid, the plasticity of the interacting domain formed by omega(6) and core1 is compromised, which may be responsible for the entropic cost. Such tight binding of the structured mutant (omega(6)) affects initiation of transcription. However, once preinitiated, the complex elongates the RNA chain efficiently. The initiation of transcription requires recognition of appropriate-factors by the core enzyme (core2: alpha(2)beta beta'omega). We found that the altered core enzyme (alpha(2)beta beta'omega(6)) with mutant omega showed a decrease in binding affinity to the sigma-factors (sigma(70), sigma(32) and sigma(38)) compared to that of the core enzyme containing native omega. In the absence of unstructured omega, the association of sigma-factors to the core is less efficient, suggesting that the flexible native omega plays a direct role in sigma-factor recruitment.