Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase alpha subunit

Abstract

The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase ␣ subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the ␣ subunit. To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3 half of E. coli rpoA, the gene encoding the ␣ subunit of RNA polymerase. We isolated a number of rpoA mutants defective in oxyR-dependent transcription activation at the E. coli katG promoter. Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the ␣ subunit. Reconstituted RNA polymerases containing the mutant ␣ subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promoters by OxyR. These results suggest that these two regions comprise the contact surfaces on the ␣ subunit for OxyR.Several lines of evidence indicate that transcription activation by DNA-binding transcription factors involves a direct protein-protein contact between those factors and RNA polymerase (1, 13). With RNA polymerases containing mutant ␣ subunits with C-terminal truncation, we have demonstrated that the C-terminal region of ␣ is involved in direct molecular communication with a group of transcription activator proteins (reviewed in reference 6). Together with other genetic, immunochemical, and biochemical evidence (15), it is proposed that the C-terminal one-third of the RNA polymerase ␣ subunit (contact site I) makes direct contact with a group of transcription activator proteins (class I activator proteins), most of which bind upstream of basic promoter elements (5, 6). Besides the role in class I activator protein-dependent transcription, recent studies indicate that the C-terminal region of the ␣ subunit is also involved in protein activator-independent transcription stimulation at several strong promoters, each containing an AT-rich sequence upstream of the Ϫ35 sequence (UP element) (14).Recently, we showed that OxyR protein, an activator protein for hydrogen peroxide-inducible genes, activates transcription initiation in vitro from the katG, ahpC, and oxyX promoters by wild-type RNA polymerase but not by mutant RNA polymerases containing a C-terminal-truncated ␣ subunit (19). Furthermore, we demonstrated that OxyR exerts cooperative binding to promoter DNA with wild-type RNA polymerase but not with the mutant RNA polymerases. These observations suggest that direct protein-protein contact between the OxyR protein and the C-terminal region of the ␣ subunit plays an essential role in transcription activation at OxyR-dependent promoters.To carry out detailed mapping of the contact site with the OxyR protein in the C-terminal contact site I region of the ␣ subunit, we isolated a number of rpoA ...

Analysis of DNA Binding and Transcriptional Activation by the LysR-Type Transcriptional Regulator CbbR of Xanthobacter flavus

“…Following activation of the regulator by the binding of a ligand, the second dimer repositions itself from IR 3 to IR 2 , resulting in the binding of the regulator adjacent to the binding site of RNA polymerase. The regulator is now properly positioned to make productive contacts with the alpha or other subunits of RNA polymerase, resulting in transcription initiation (21,28). Like OccR and OxyR, CbbR introduces a bend in the DNA, which is relaxed following the binding of the ligand (27).…”

Peroxide‐Sensing Transcriptional Regulators in Bacteria

“…C199-SOH rapidly reacts with C208-SH to form an intramolecular disulfide bond (65). This induces structural changes in the regulatory domain (amino acids 80 to 305) (18,65) that result in altered associations between the subunits within the tetramer, leading to altered DNA binding properties and allowing productive interaction between OxyR and RNA polymerase (18,106,108).…”

“…While OxyR is primarily thought of as a transcriptional activator under oxidizing conditions that acts through direct interaction with the RNA polymerase ␣ subunit (61,103,106), OxyR can function as either a repressor or activator under both oxidizing and reducing conditions (19,45,49,96,108,121).…”

Importance of Tetramer Formation by the Nitrogen Assimilation Control Protein for Strong Repression of Glutamate Dehydrogenase Formation in Klebsiella pneumoniae

“…At the hut and put operons, NAC activates transcription by a mechanism that requires NAC binding to a site centered at Ϫ64 relative to the start of transcription (15). Data from other LysR-type regulators suggest by analogy that NAC may interact with the ␣-subunit of RNA polymerase in such cases (22,38). At the dad, ure, and cod operons, NAC activates transcription by a mechanism that requires binding to a site centered at Ϫ44, Ϫ49, and Ϫ59, respectively (20, 29; unpublished data).…”