The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription.
Bacteriophage T4 middle promoters, which are transcribed using phage-modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host sigma 70 consensus sequence at -10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at -30. While the T4 middle promoter PuvsX has these -10 and -30 motifs, it also has matches to the MotA box at -35, -51, -70, and -87. We show that MotA binds to PuvsX DNA, footprinting a region that includes the MotA boxes at -30, -35, and -51. Very high levels of MotA are required for footprinting and gel-shift experiments, and protein-DNA complexes formed in the presence of both phage-modified polymerase and MotA are more resistant to HindIII cleavage than those formed with either protein alone. These results suggest that MotA-DNA interactions may be stabilized by phage-modified polymerase. Sequences between -18 and -38 are absolutely required for MotA activation of transcription, but sequences upstream of -38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect PuvsX into a core promoter, downstream of -38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein-DNA interactions.
March-Amegadzie
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Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hinton
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DM
D017122:Bacteriophage T4 / Q000235:genetics*; D001483:Base Sequence; D002712:Chlorides / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D018698:Glutamic Acid / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012318:RNA Polymerase I / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
D002712:Chlorides; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C067996:MotA protein, Enterobacteria phage T4; D012333:RNA, Messenger; D014157:Transcription Factors; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; D018698:Glutamic Acid; D012318:RNA Polymerase I