Substitutions in region 2.4 of sigma70 allow recognition of the sigmaS-dependent aidB promoter.
The strict dependence of transcription from the aidB promoter (PaidB) on the Esigma(S) form of RNA polymerase is because of the presence of a C nucleotide as the first residue of the -10 promoter sequence (-12C), which does not allow an open complex formation by Esigma(70). In this report, sigma(70) mutants carrying either the Q437H or the T440I single amino acid substitutions, which allow -12C recognition by sigma(70), were tested for their ability to carry out transcription from PaidB. The Gln-437 and Thr-440 residues are located in region 2.4 of sigma(70) and correspond to Gln-152 and Glu-155 in sigma(S). Interestingly, the Q437H mutant of sigma(70), but not T440I, was able to promote an open complex formation and to initiate transcription at PaidB. In contrast to T440I, a T440E mutant was proficient in carrying out transcription from PaidB. No sigma(70) mutant displayed significantly increased interaction with a PaidB mutant in which the -12C was substituted by a T (PaidB((C12T))), which is also efficiently recognized by wild type sigma(70). The effect of the T440E mutation suggests that the corresponding Glu-155 residue in sigma(S) might be involved in -12C recognition. However, substitution to alanine of the Glu-155 residue, as well as of Gln-152, in the sigma(S) protein did not significantly affect Esigma(S) interaction with PaidB. Our results reiterate the importance of the -12C residue for sigma(S)-specific promoter recognition and strongly suggest that interaction with the -10 sequence and open complex formation are carried out by different determinants in the two sigma factors.
Lacour
Stephan
S
Swiss Federal Institute of Environmental Technology (EAWAG), Uberlandstrasse 133, CH-8600 Dübendorf, Switzerland.
Leroy
Olivier
O
Kolb
Annie
A
Landini
Paolo
P
D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000235:genetics*; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011196:Potassium Permanganate / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D014158:Transcription, Genetic
C090096:AidB protein, E coli; D029968:Escherichia coli Proteins; D012808:Sigma Factor; D011196:Potassium Permanganate; D005973:Glutamine; D013912:Threonine; D018698:Glutamic Acid; D006493:Heparin; D004247:DNA; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I