Purified MotA protein binds the -30 region of a bacteriophage T4 middle-mode promoter and activates transcription in vitro.
The bacteriophage T4-encoded MotA protein is critical for transcription from T4 middle-mode promoters. However, a direct interaction of this protein with a middle-mode promoter has not previously been demonstrated. We have cloned the motA gene and overexpressed the gene product using the T7 expression system. A simple procedure was then developed to purify the MotA protein to homogeneity. Using the purified protein we have demonstrated that MotA protein binds to the -30 region of the middle-mode promoter PuvsY. This promoter has previously been shown to be a necessary component of a T4 replication origin, and thus MotA is also a T4 origin-binding protein. Modified RNA polymerase purified from T4-infected cells was used to establish middle-mode transcription in vitro. Transcription from PuvsY was markedly enhanced by the addition of MotA protein, whether or not the template contained the cytosine modifications characteristic of T4 DNA. However, transcription from PuvsY was apparently independent of the MotA protein when unmodified RNA polymerase from uninfected cells was used.
Schmidt
R P
RP
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Kreuzer
K N
KN
D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; C067996:MotA protein, Enterobacteria phage T4; D014157:Transcription Factors; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases