A novel selection was developed for RpoA α-CTD mutants altered in activation by the TyrR regulatory protein of
E. coli
K-12. This allowed the identification of an aspartate to asparagine substitution in residue 250 (DN250) as an Act
-
mutation. Amino acid residues known to be close to D250 were altered by
in vitro
mutagenesis, and substitutions DR250, RE310 and RD310 were all shown to be defective in activation. None of these mutations caused defects in UP regulation. The
rpoA
mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. 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, 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 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 αCTD 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.
Activation and Repression at the Escherichia coli ynfEFGHI Operon Promoter
