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Validation of Omega Subunit of RNA Polymerase as a Functional Entity.

ABSTRACT: The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (?2??'? ?) containing the smallest subunit, ?. 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, ?', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ? and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ?) 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 ? during transcription, several dominant lethal mutants of ? were isolated. The mutants showed higher binding affinity compared to that of native ? to the ?2??' subassembly. We observed that the interaction between ?2??' 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 ? having either point mutation or silent in nature are more structured in comparison to the native ?. 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 ? makes it more rigid, which affects the plasticity of the interacting domain formed by ? and ?2??'. Here, we attempted to describe how the conformational flexibility of the ? helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ? has a suppressor mapped near the catalytic center of the ?' subunit, and it is the same for both types of mutants.

SUBMITTER: Patel UR

PROVIDER: S-EPMC7700224 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Validation of Omega Subunit of RNA Polymerase as a Functional Entity.

Patel Unnatiben Rajeshbhai UR Gautam Sudhanshu S Chatterji Dipankar D

Biomolecules 20201123 11

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, <i>E. col ...[more]

PMID: 33238579

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Project description:The catalytic core of the RNA polymerase of most eubacteria is composed of two M-NM-1 subunits and M-NM-2, M-NM-2M-bM-^@M-^Y and M-OM-^I subunits. In Escherichia coli, the M-OM-^I subunit (encoded by the rpoZ gene) has been suggested to assist M-NM-2M-bM-^@M-^Y during RNA polymerase core assembly. The function of the M-OM-^I subunit is particularly interesting in cyanobacteria because the cyanobacterial M-NM-2M-bM-^@M-^Y is split to N-terminal M-NM-3 and C-terminal M-NM-2M-bM-^@M-^Y subunits. The M-bM-^HM-^FrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions although the mutant cells showed low light-saturated photosynthetic activity, low Rubisco content and accumulated high quantities of protective carotenoids and M-NM-1-tocopherol. The M-bM-^HM-^FrpoZ strain contained 15% less of the primary M-OM-^C factor, SigA, than the control strain, and recruitment of SigA to the RNA polymerase core was inefficient in M-bM-^HM-^FrpoZ. Thus, a cyanobacterial RNA polymerase holoenzyme lacking the M-OM-^I subunit contains less frequently the primary M-OM-^C factor. A DNA microarray analysis revealed that this leads to specific down-regulation of highly expressed genes, like genes encoding subunits for Rubisco, ATP synthase, NADH-dehydrogenase and carbon concentrating mechanisms. On the contrary, many genes showing only low or moderate expression in the control strain were up-regulated in M-bM-^HM-^FrpoZ. A conserved -10 region was detected in promoters showing up or down-regulation in M-bM-^HM-^FrpoZ, but -35 regions of down-regulated genes completely differed from -35 regions of up-regulated genes. Cells from cyanobacteria Synechocystis sp. PCC 6803 named as control strain (CS) and RNA polymerase omega subunit inactivation strain, M-NM-^TrpoZ, were harvested (A730=1, 40 mL) directly from standard growth conditions (continuous illumination at the PPFD of 40 M-BM-5mol m-2s-1, 32M-BM-0C, ambient CO2). From three to four independent experiments were performed at each conditions.

Project description:In growing bacterial cells, the global reorganization of transcription is associated with alterations of RNA polymerase composition and the superhelical density of the DNA. However, the existence of any regulatory device coordinating these changes remains elusive. Here we show that in an exponentially growing Escherichia coli rpoZ mutant lacking the polymerase ? subunit, the impact of the E?(38) holoenzyme on transcription is enhanced in parallel with overall DNA relaxation. Conversely, overproduction of ?(70) in an rpoZ mutant increases both overall DNA supercoiling and the transcription of genes utilizing high negative superhelicity. We further show that transcription driven by the E?(38) and E?(70) holoenzymes from cognate promoters induces distinct superhelical densities of plasmid DNA in vivo. We thus demonstrate a tight coupling between polymerase holoenzyme composition and the supercoiling regimen of genomic transcription. Accordingly, we identify functional clusters of genes with distinct ? factor and supercoiling preferences arranging alternative transcription programs sustaining bacterial exponential growth. We propose that structural coupling between DNA topology and holoenzyme composition provides a basic regulatory device for coordinating genome-wide transcription during bacterial growth and adaptation. IMPORTANCE Understanding the mechanisms of coordinated gene expression is pivotal for developing knowledge-based approaches to manipulating bacterial physiology, which is a problem of central importance for applications of biotechnology and medicine. This study explores the relationships between variations in the composition of the transcription machinery and chromosomal DNA topology and suggests a tight interdependence of these two variables as the major coordinating principle of gene regulation. The proposed structural coupling between the transcription machinery and DNA topology has evolutionary implications and suggests a new methodology for studying concerted alterations of gene expression during normal and pathogenic growth both in bacteria and in higher organisms.

Dataset Information

Validation of Omega Subunit of RNA Polymerase as a Functional Entity.

Publications

Validation of Omega Subunit of RNA Polymerase as a Functional Entity.

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