New roles for conserved regions within a sigma 54-dependent enhancer-binding protein.
23 amino acid substitutions were made in the C7 and C3 regions of pspFDeltaHTH, a protein required to convert sigma(54) closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with sigma(54) and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (, ). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with sigma(54) during the transition state but not important in inducing sigma(54) holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation.
Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095-1569, USA.
Chih M
CM
Lew
Jay D
JD
Gralla
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004742:Enhancer Elements, Genetic; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000502:physiology*; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C098954:PspF protein, E coli; D012808:Sigma Factor; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D004247:DNA