A promoter melting region in the primary sigma factor of Bacillus subtilis. Identification of functionally important aromatic amino acids.
Sigma factor (sigma) is a dissociable subunit of bacterial RNA polymerase that determines promoter recognition. It has been proposed that a cluster of highly conserved aromatic amino acids in bacterial sigma factors (region 2.3) defines a melting motif that functions in strand-separation during open complex formation. We demonstrate that many alterations in region 2.3 of the Bacillus subtilis sigma A protein specifically impair open complex formation. The region 2.3 mutations can be grouped in three classes: (1) mutations that do not significantly affect promoter recognition or melting; (2) mutations that lead to cold-sensitive transcription of linear templates; and (3) mutations that lead to little activity on linear templates but retain activity at high temperatures on supercoiled templates. RNA polymerase holoenzymes containing sigma factor melting mutants (classes 2 and 3) form predominantly closed complexes at 40 degrees C and are defective for RNA synthesis when initiation is rate-limiting. The melting defect of these mutant sigma factors is suppressed by template supercoiling, but further enhanced by inclusion of the auxiliary RNA polymerase subunit delta. Consequently, in the presence of the delta polypeptide, the mutant holoenzymes display cold-sensitive transcription on supercoiled templates: conditions which mimic the in vivo situation. A subset of these mutations also affects promoter selectivity, suggesting that region 2.3 may participate in both -10 recognition and DNA melting.
Juang
Y L
YL
Section of Microbiology, Cornell University, Ithaca, NY 14853.
Helmann
J D
JD
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism; D003850:Deoxyribonuclease I; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I