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 (ω), which is predominantly α-helical, in contrast to largely unstructured native ω, and then studied its assembly with reconstituted core1 (αββ') by a biophysical approach. The mutant showed higher binding affinity compared to native ω. We observed that the interaction between core1 and ω is driven by highly negative enthalpy and a small but unfavorable negative entropy term. Extensive structural alteration in ω makes it more rigid, the plasticity of the interacting domain formed by ω and core1 is compromised, which may be responsible for the entropic cost. Such tight binding of the structured mutant (ω) 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: αββ'ω). We found that the altered core enzyme (αββ'ω) with mutant ω showed a decrease in binding affinity to the σ-factors (σ, σ and σ) 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.

The role of ω‐subunit of Escherichia coli RNA polymerase in stress response

“…We had generated a dominant‐negative mutant for ω (ω 6 ), which is predominantly α‐helical, and when incorporated into holo RNAP, the enzyme is initiation defective. In a recent work from our laboratory, the thermodynamic cost guiding the plasticity in β′‐ω interaction for RNAP assembly and sigma factor recognition have been quantified (Bhowmik, Bhardwaj, & Chatterji, ). Thus, in this report, we are inclined to quantitate the binding parameters of ppGpp to E. coli RNAP as a function of the plasticity in the structure of ω.…”

“…The disordered nature of ω is important for maintaining the plasticity of the enzyme during transcription initiation and binding of sigma factors (Bhowmik et al., ; Sarkar et al., ). We wanted to see whether unstructured nature of ω is required for binding of ppGpp to RNAP.…”

“…Growth profile was analyzed following a starvation period of (a) 0 hr, (b) 6 hr and (c) 24 hr. (d) Measurement of growth recovery of wildtype (WT), ∆rpoZ, ∆rpoZ pBAD-rpoZ and ∆relA/∆spoT strains strain by colony-forming unit (CFU) count after a starvation period of 24 hr | 363 The disordered nature of ω is important for maintaining the plasticity of the enzyme during transcription initiation and binding of sigma factors (Bhowmik et al, 2017;Sarkar et al, 2013). We wanted to see whether unstructured nature of ω is required for binding of ppGpp to RNAP.…”

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome

“…These findings indicate the direct molecular contact between RpoZ and other subunits of RNAP. Several lines of observation indicated the involvement of RpoZ in the functional control of RNAP ( 14 , 16 ). A functional link between the RpoZ subunit and the stringent response has been elucidated: first, in vitro transcription by purified RNAP without RpoZ subunit was found to be insensitive to ppGpp ( 17 ), and the RpoZ-less RNAP regains its sensitivity to (p)ppGpp upon the external addition of the RpoZ subunit ( 18 ) or the protein DksA, a collaborative player in the stringent response ( 19 ).…”

On an algorithmic definition for the components of the minimal cell

“…229 As an example of the algebraic approach to represent the synthesis of a cellular 230 element let's take the enzyme RNA polymerase, which will be abbreviated here as 'pol'. 231 For this simplified illustration we will consider that pol is constituted only by α, β and 232 β subunits, ignoring the important σ factor [61], but considering the ω subunit [62], 233 thus we consider pol = 2αββ ω, because there are two α subunits in this enzyme. Now 234 we can substitute in the expression for pol the 2α part by the corresponding binary 235 operator α, α , because we know that α, α ⇒ 2α, and so on, until we decompose pol 236 into their subunits, say 237 pol = 2α, ββ ω…”