Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions.
According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the alpha subunit's C-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of sigma(70) (sigma(70) R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these sigma(70) R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and sigma(70) R4 can interact in solution (i.e., "off-DNA"). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of sigma(70) R4 that reduce activation in E. coli also reduce the SoxS-sigma(70) R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur "on-DNA" at class II promoters. Finding that SoxS contacts sigma(70) R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein-protein contacts with two different subunits of RNAP.
Zafar
M Ammar
MA
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Shah
Ishita M
IM
Wolf
Richard E
RE
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D001120:Arginine / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism* / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004843:Epistasis, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D030781:Organisms, Genetically Modified; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D025941:Protein Interaction Mapping; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C070725:SoxS protein, E coli; D001120:Arginine; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D000409:Alanine