Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides.
Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma factors. Of these, sigma(F) is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma(F) (E-sigma(F)), and only two genes of known function are exclusively under the control of E-sigma(F), spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma(F), we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma(F) in vivo. The results indicate a weak sigma(F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma factors, as well as to sigma(A). There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma factors. The looseness of the sigma(F) promoter requirement contrasts with the strict requirement for sigma(A)-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma(F) in vivo.
Amaya
E
E
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Khvorova
A
A
Piggot
P J
PJ
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016384:Consensus Sequence; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D009841:Oligonucleotides; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D001616:beta-Galactosidase