An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription.
The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters. MotA alone binds to the -30 region of T4 middle promoters, a region that contains the MotA box consensus sequence [(t/a)(t/a)TGCTT(t/c)A]. We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons. In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P(uvsX) similarly, and the proteins yield similar footprints on P(uvsX). However, Mot21 is severely defective in the activation of transcription. On native protein gels, a new protein species is seen after incubation of the sigma70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and sigma70. Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.
Gerber
J S
JS
Section on Nucleic Acid Biochemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Hinton
D M
DM
D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C067996:MotA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014157:Transcription Factors; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases