Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: involvement of the T4 MotA activator and the T4 AsiA protein, a sigma 70 binding protein, in the formation of the open complex.
Bacteriophage T4 middle promoters have excellent matches to the -10 consensus sequence for the sigma 70 subunit of Escherichia coli RNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the sigma 70 -35 consensus. E. Coli RNA polymerase transcribes from middle promoters with or without the activator. In contrast, transcription by T4-modified E. coli RNA polymerase, which is present during T4 infection, requires NotA. We show that transcription by unmodified polymerase from the T4 middle promoter P uvsx is independent of the specific sequences within the -35 region, and the Dnase I footprint obtained with unmodified polymerase and P uvsx resembles those seen previously with E. coli extended -10" promoters. In contrast, although T4-modified polymerase alond binds P uvsx, promoter unwinding and detection of a Dnase I footprint requires MotA. This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position -20. Previous work has indicated that the T4 AsiA protein, which binds tightly to sigma 70, is the phage modification required for MotA activation. We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, Dnase I protection of P uvsx is now similar to that obtained with the fully modified polymerase and MotA up to around position -40. However, protection upstream of -40 is still similar to that seen with unmodified polymerase. Our results support the idea that MotA-dependent activation requires AsiA binding to sigma 70 to achieve specific protein-DNA contacts within the -20 to -40 region of a middle promoter.
Hinton
D M
DM
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
March-Amegadzie
R
R
Gerber
J S
JS
Sharma
M
M
D017122:Bacteriophage T4 / Q000235:genetics*; D001483:Base Sequence; D018983:DNA Footprinting; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C067996:MotA protein, Enterobacteria phage T4; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I