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The interaction of ?2 with the RNA polymerase ?' subunit functions as an activation to repression switch.

ABSTRACT: The ? gene is encoded in broad-host range and low-copy plasmids. It is genetically linked to antibiotic resistance genes of the major human pathogens of phylum Firmicutes. The homodimeric forms of ? (?2) coordinate the plasmid copy number control, faithful partition (?2 and ?2) and better-than-random segregation (??2?) systems. The promoter (P) of the ??? operon (P?) transiently interacts with ?2. Adding ?2 facilitates the formation of stable ?2·P? complexes. Here we show that limiting ?2 interacts with the N-terminal domain of the ?' subunit of the Bacillus subtilis RNA polymerase (RNAP-?(A)) vegetative holoenzyme. In this way ?2 recruits RNAP-?(A) onto P? DNA. Partial P? occupancy by ?2 increases the rate at which RNAP-?(A) complex shifts from its closed (RPC) to open (RPO) form. This shift increases transcription activation. Adding ?2 further increases the rate of P? transcription initiation, perhaps by stabilizing the ?2·P? complex. In contrast, full operator occupancy by ?2 facilitates RPC formation, but it blocks RPO isomerization and represses P? utilization. The stimulation and inhibition of RPO formation is the mechanism whereby ?2 mediates copy number fluctuation and stable plasmid segregation. By this mechanism, ?2 also indirectly influences the acquisition of antibiotic resistance genes.

SUBMITTER: Volante A

PROVIDER: S-EPMC4627068 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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The interaction of ω2 with the RNA polymerase β' subunit functions as an activation to repression switch.

Volante Andrea A Carrasco Begoña B Tabone Mariangela M Alonso Juan C JC

Nucleic acids research 20150803 19

The ω gene is encoded in broad-host range and low-copy plasmids. It is genetically linked to antibiotic resistance genes of the major human pathogens of phylum Firmicutes. The homodimeric forms of ω (ω2) coordinate the plasmid copy number control, faithful partition (ω2 and δ2) and better-than-random segregation (ζϵ2ζ) systems. The promoter (P) of the ωϵζ operon (Pω) transiently interacts with ω2. Adding δ2 facilitates the formation of stable ω2·Pω complexes. Here we show that limiting ω2 intera ...[more]

PMID: 26243774

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Project description:A novel selection was developed for mutants of the C-terminal domain of RpoA (α-CTD) altered in activation by the TyrR regulatory protein of Escherichia coli K-12. This allowed the identification of an aspartate to asparagine substitution at residue 250 (DN250) as an activation-defective (Act-) mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and the substitutions DR250, RE310, and RD310 were all shown to be defective in activation. None of these mutations caused defects in regulation of the upstream promoter (UP) element. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. The TyrR mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77, and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, and EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act- tyrR mutants were isolated in an rpoA+ strain, adding positions R77, D97, K101, D118, R119, R121, and E141 to known residues S95 and D103 and defining the activation patch on the amino-terminal domain (NTD) of TyrR. These results support a model for activation of TyrR-regulated genes where the activation patch on the TyrR NTD interacts with the TyrR-specific patch on the α-CTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants as activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene tyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore, TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence and important plant interactions.

Dataset Information

The interaction of ?2 with the RNA polymerase ?' subunit functions as an activation to repression switch.

Publications

The interaction of ω2 with the RNA polymerase β' subunit functions as an activation to repression switch.

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