Roles for the C-terminal region of sigma 54 in transcriptional silencing and DNA binding.
Twenty-one conserved positively charged and aromatic amino acids between residues 331 and 462 of sigma 54 were changed to alanine, and the mutant proteins were studied by transcription, band shift analysis, and footprinting in vitro. A small segment corresponding to the rpoN box was found to be most important for binding duplex DNA. Two amino acids, 52 residues apart, were found to be critical for maintaining transcriptional silencing in the absence of activator. These two activator bypass mutants and several other mutants failed to bind the type of fork junction DNA thought to be required to maintain silencing. The two bypass mutants showed a binding pattern to DNA probes that was unique, both in comparison to other C-terminal mutants and to previously known N-terminal bypass mutants. On this basis, a model is proposed for the role of the C terminus and the N terminus of sigma 54 in enhancer-dependent transcription.
Wang
L
L
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095-1569, USA.
Gralla
J D
JD
D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D012808:Sigma Factor; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54