Activation by tyrr in escherichia coli k-12 by interaction between tyrr and the a-subunit of rna polymerase

Helen Camakaris, Ji Yang, Tadashi Fujii, James Pittard

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

A novel selection was developed for mutants of the C-terminal domain of RpoA (a-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 (Act2) 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 Act2 tyrR mutants were isolated in an rpoA1 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 a-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.

Original languageEnglish
Article numbere00252-21
JournalJournal of Bacteriology
Volume203
Issue number19
DOIs
Publication statusPublished - 10-2021
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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