Dataset Information

Altered oligomerization properties of N316 mutants of Escherichia coli TyrR.

ABSTRACT: The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrR(E274Q), a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrR(N316D) and TyrR(N316R) mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrR(N316D) and TyrR(N316R), respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrR(N316D).

SUBMITTER: Koyanagi T

PROVIDER: S-EPMC2593242 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Altered oligomerization properties of N316 mutants of Escherichia coli TyrR.

Koyanagi Takashi T Katayama Takane T Suzuki Hideyuki H Kumagai Hidehiko H

Journal of bacteriology 20081017 24

The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrR(E274Q), a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrR(N316D) and TyrR(N316R) mutants. Gel filtration analyses reveal ...[more]

PMID: 18931124

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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

Altered oligomerization properties of N316 mutants of Escherichia coli TyrR.

Publications

Altered oligomerization properties of N316 mutants of Escherichia coli TyrR.

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