Abstract
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ’ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β’, and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ’ subassembly. We observed that the interaction between α2ββ’ and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code’s position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ’. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β’ subunit, and it is the same for both types of mutants.
Highlights
A few decades ago, Tom Silhavy proposed that the α-subunit of E. coli RNA polymerase was the “Cinderella”-subunit, often ignored as useless little stepsister by larger subunits [1]
Transcription is the first step of gene expression, which starts with the synthesis of RNA from DNA by the RNA polymerase (RNAP) enzyme
One such study was the development of a protocol by Burgess and Jendrisak to purify bacterial RNA polymerase using Polymin P precipitation and DNA-cellulose chromatography [12]
Summary
A few decades ago, Tom Silhavy proposed that the α-subunit of E. coli RNA polymerase was the “Cinderella”-subunit, often ignored as useless little stepsister by larger subunits [1]. The ubiquitous role of α-subunit in transcription regulation soon became apparent, and the name of a useless little sister was no more applicable. This review is aimed at suggesting that the smallest subunit ω received step-sisterly treatment from others, but it is getting clear that ω has an important function in transcription biology
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