Identifying N60D mutation in ω subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach

Sabareesh, Varatharajan; Sarkar, Paramita; Sardesai, Abhijit A.; Chatterji, Dipankar

doi:10.1039/c0an00130a

Cited by 6 publications

(6 citation statements)

References 35 publications

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“…3A) bearing N60D alteration results in an increment in molecular mass of by 0.98 -1 Da that we have tested and verified in an independent study involving mass spectrometry (MS) (51). MS analysis also ensured that the isolated 6 was 100% mutant variety with no detectable contamination of the wild-type (51).…”

Section: Isolation Of Dominant Lethal Variants and Rationale Of Thementioning

confidence: 96%

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

Sarkar

Sardesai

Murakami

et al. 2013

Journal of Biological Chemistry

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Background: Although universally conserved, is the only nonessential subunit of RNA polymerase, and its function is mostly undetermined. Results: Dominant lethal mutants of were isolated and analyzed genetically and biochemically. Conclusion: Mutations in that alter its structure interfere with RNA polymerase catalytic properties. Significance: Results suggest a critical role for during transcription, bypassing the effects of compensatory proteins.

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Section: Isolation Of Dominant Lethal Variants and Rationale Of Thementioning

confidence: 96%

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

Sarkar

Sardesai

Murakami

et al. 2013

Journal of Biological Chemistry

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“…In this work, we purified the wild-type u and its dominant negative variant, u 6 (13,21), and then studied their assembly with reconstituted RNAP (core1: a 2 bb 0 ). Subsequently the interaction of s-factors with reconstituted RNAP (core2: a 2 bb 0 u; mutated core2: a 2 bb 0 u 6 ) was also analyzed.…”

Section: Introductionmentioning

confidence: 99%

Influence of Flexible “ ω ” on the Activity of E. coli RNA Polymerase: A Thermodynamic Analysis

Bhowmik

Bhardwaj

Chatterji

2017

Biophysical Journal

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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.

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“…As mentioned before, the silent mutant ω has the same sequence of amino acids as that of wild-type protein, however, with a third base change in the code for alanine at the 82nd position. The sequence of the mutant ω, having point mutation or silent in nature, was firmly established by mass spectroscopy-based sequence analysis [ 37 ]. The silent mutant has a folded conformation, despite having a wild-type sequence (ω9, Figure 2 A) and represents a classic case of two conformation of the same protein due to codon degeneracy.…”

Section: Functions Of ω Subunit In Rnapmentioning

confidence: 99%

Validation of Omega Subunit of RNA Polymerase as a Functional Entity

2020

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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.

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Identifying N60D mutation in ω subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach

Cited by 6 publications

References 35 publications

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

Influence of Flexible “ ω ” on the Activity of E. coli RNA Polymerase: A Thermodynamic Analysis

Validation of Omega Subunit of RNA Polymerase as a Functional Entity

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