N- and C-terminal regions of the quorum-sensing activator TraR cooperate in interactions with the alpha and sigma-70 components of RNA polymerase.
Positive control (PC) mutants defining 20 residues of the quorum-sensing activator TraR were isolated that bind DNA but show defects in activating transcription from class I, class II or both types of promoters. These PC residues, located in both the N- and C-terminal regions, combine to form three patches, one on the top (II) and two near the DNA binding domain on both lateral faces of the dimer (I and III). Patches I and II, but not patch III, involve residues from both protomers and are essential for activation. TraR-mediated activation in Escherichia coli requires expression of the alpha-subunit of Agrobacterium (alpha(At)). We report that TraR also activates a class II promoter in E. coli when coexpressed with sigma(70)(At). Analyses in E. coli expressing alpha(At), sigma(70)(At) or both subunits indicate that most of the PC residues are important for interactions with alpha(At) and that these interactions are predominant for activation of class II promoters. Using the E. coli system we identified nine residues in the C-terminal domain of alpha(At) that are required for stimulating TraR-mediated activation. We conclude that N- and C-terminal residues of TraR from both protomers cooperate to define regions of the protein important for interactions with RNAP.
Qin
Yinping
Y
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Keenan
Carrie
C
Farrand
Stephen K
SK
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D053038:Quorum Sensing; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D029968:Escherichia coli Proteins; D012808:Sigma Factor; C535057:TraR protein, E coli; D014157:Transcription Factors; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases