title: Influence of Flexible "ω" on the Activity of E. coli RNA Polymerase: A Thermodynamic Analysis.
abstract: The Escherichia coli RNA polymerase (RNAP) is a multisubunit protein complex containing the smallest subunit, ω. Despite the evolutionary conservation of ω and its role in assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) 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 ω, we isolated a dominant negative mutant of ω (ω6), which is predominantly α-helical, in contrast to largely unstructured native ω, and then studied its assembly with reconstituted core1 (α2ββ') by a biophysical approach. The mutant showed higher binding affinity compared to native ω. We observed that the interaction between core1 and ω6is driven by highly negative enthalpy and a small but unfavorable negative entropy term. Extensive structural alteration in ω6makes it more rigid, the plasticity of the interacting domain formed by ω6and core1 is compromised, which may be responsible for the entropic cost. Such tight binding of the structured mutant (ω6) affects initiation of transcription. However, once preinitiated, the complex elongates the RNA chain efficiently. The initiation of transcription requires recognition of appropriate σ-factors by the core enzyme (core2: α2ββ'ω). We found that the altered core enzyme (α2ββ'ω6) with mutant ω showed a decrease in binding affinity to the σ-factors (σ70, σ32and σ38) compared to that of the core enzyme containing native ω. In the absence of unstructured ω, the association of σ-factors to the core is less efficient, suggesting that the flexible native ω plays a direct role in σ-factor recruitment.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Debipreeta
authors.initials: D
authors.lastname: Bhowmik
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Neerupma
authors.initials: N
authors.lastname: Bhardwaj
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. Electronic address: dipankar@mbu.iisc.ernet.in.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chatterji
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D021122:Protein Subunits; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases