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The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit.

ABSTRACT: Gene transfer agents (GTAs) are small virus-like particles that indiscriminately package and transfer any DNA present in their host cell, with clear implications for bacterial evolution. The first transcriptional regulator that directly controls GTA expression, GafA, was recently discovered, but its mechanism of action has remained elusive. Here, we demonstrate that GafA controls GTA gene expression via direct interaction with the RNA polymerase omega subunit (Rpo-ω) and also positively autoregulates its own expression by an Rpo-ω-independent mechanism. We show that GafA is a modular protein with distinct DNA and protein binding domains. The functional domains we observe in Rhodobacter GafA also correspond to two-gene operons in Hyphomicrobiales pathogens. These data allow us to produce the most complete regulatory model for a GTA and point toward an atypical mechanism for RNA polymerase recruitment and specific transcriptional activation in the Alphaproteobacteria.

SUBMITTER: Sherlock D

PROVIDER: S-EPMC9638019 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit.

Sherlock David D Fogg Paul C M PCM

Cell reports 20220801 6

Gene transfer agents (GTAs) are small virus-like particles that indiscriminately package and transfer any DNA present in their host cell, with clear implications for bacterial evolution. The first transcriptional regulator that directly controls GTA expression, GafA, was recently discovered, but its mechanism of action has remained elusive. Here, we demonstrate that GafA controls GTA gene expression via direct interaction with the RNA polymerase omega subunit (Rpo-ω) and also positively autoregu ...[more]

PMID: 35947951

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Project description: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.

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.

Dataset Information

The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit.

Publications

The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit.

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