A sigma32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity.
sigma32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of sigma32 with other sigma factors in the sigma70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant sigma32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of sigma70, which competes with sigma32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.
Joo
D M
DM
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Ng
N
N
Calendar
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D012321:DNA-Directed RNA Polymerases; C063842:dnaK protein, E coli