Dataset Information

Unraveling the Role of Silent Mutation in the ?-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription.

ABSTRACT: The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, ?2??'??. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ?-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the mutant was shown to render the RNA polymerase defective in transcription initiation (?6, N60D) and gave an insight on the function of ? in RNA polymerase. Serendipitously, we also obtained a silent mutant, and the mutant was found to be lethal during the isolation of toxic mutants. The primary focus of this study is to understand the mechanistic details of this lethality. Isolated ? shows a predominantly unstructured circular dichroism profile and becomes ?-helical in the enzyme complex. This structural transition is perhaps the reason for this lack of function. Subsequently, we generated several silent mutants of ? to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ? is transcriptionally inactive. The CodonPlus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

SUBMITTER: Patel UR

PROVIDER: S-EPMC6822122 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Unraveling the Role of Silent Mutation in the ω-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription.

Patel Unnatiben Rajeshbhai UR Gautam Sudhanshu S Chatterji Dipankar D

ACS omega 20191014 18

The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, α2ββ'σω. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ω-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the ...[more]

PMID: 31681877

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Project description:BACKGROUND:An issue associated with efficient bioethanol production is the fact that the desired product is toxic to the biocatalyst. Among other effects, ethanol has previously been found to influence the membrane of E. coli in a dose-dependent manner and induce changes in the lipid composition of the plasma membrane. We describe here the characterization of a collection of ethanol-tolerant strains derived from the ethanologenic Escherichia coli strain FBR5. RESULTS:Membrane permeability assays indicate that many of the strains in the collection have alterations in membrane permeability and/or responsiveness of the membrane to environmental changes such as temperature shifts or ethanol exposure. However, analysis of the strains by gas chromatography and mass spectrometry revealed no qualitative changes in the acyl chain composition of membrane lipids in response to ethanol or temperature. To determine whether these strains contain any mutations that might contribute to ethanol tolerance or changes in membrane permeability, we sequenced the entire genome of each strain. Unexpectedly, none of the strains displayed mutations in genes known to control membrane lipid synthesis, and a few strains carried no mutations at all. Interestingly, we found that four independently-isolated strains acquired an identical C → A (V244 V) silent mutation in the ferric citrate transporter gene fecA. Further, we demonstrated that either a deletion of fecA or over-expression of fecA can confer increased ethanol survival, suggesting that any misregulation of fecA expression affects the cellular response to ethanol. CONCLUSIONS:The fact that no mutations were observed in several ethanol-tolerant strains suggested that epigenetic mechanisms play a role in E. coli ethanol tolerance and membrane permeability. Our data also represent the first direct phenotypic evidence that the fecA gene plays a role in ethanol tolerance. We propose that the recurring silent mutation may exert an effect on phenotype by altering RNA-mediated regulation of fecA expression.

Dataset Information

Unraveling the Role of Silent Mutation in the ?-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription.

Publications

Unraveling the Role of Silent Mutation in the ω-Subunit of <i>Escherichia coli</i> RNA Polymerase: Structure Transition Inhibits Transcription.

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