Abstract
The bacteriophage T4 motA protein is required for transcription from T4 middle promoters. These promoters, which contain the Escherichia coli promoter consensus sequence at the -10 region (TATAAT) but a unique sequence centered at -30 ((a/t)(a/t)TGCTT(t/c)A) (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258), become active about 2 min after infection, a time when the host RNA polymerase has been modified by phage proteins. This paper shows that motA protein binds to a T4 middle promoter in vitro and that the addition of the motA protein allows in vitro transcription from this promoter by T4-modified RNA polymerase. The T4 motA gene was cloned into a multicopy plasmid that complemented T4 motA mutants in vivo. MotA protein, partially purified from cells containing a motA+ plasmid, specifically retarded the electrophoretic mobility of an oligomer containing the T4 middle promoter located 195 bases upstream of uvsX (PuvsX). RNA polymerase isolated from infected cells during T4 middle gene expression supported in vitro transcription from PuvsX only when fractions containing the motA protein were added. In contrast, unmodified host RNA polymerase catalyzed the synthesis of minor amounts of RNA from PuvsX, but this synthesis was not motA dependent. Thus, the in vitro transcription system described here provides the basis for a detailed study of the phage and host factors needed to regulate T4 middle gene expression.
Highlights
From the Section on Nucleic Acid Biochemistry, Laboratoroyf Biochemical Pharmacology, National Institute of Diabetes and Digestiue and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
The bacteriophage T4 motA protein is required for share sequence features with the bacterial consensus promoter transcription from T4 middle promoters
This paper shows that motA protein binds to a T4 middlepromoter in vitroand that the addition of the motA protein allows in vitro transcription from this promoter by T4-modified RNA poinfection
Summary
Po1-r.s. MotA-dependent Transcription inVitro Using RNA Polymerase from T4-infected Cells-The 1392-bp EcoRI to AsuII fragment from pDH428 (positions 1-1392) includes the assigned motA promoters PX., (position 400) and Puvsx(position. Decreasing the middle polymerase to 0.15pg did not change the effect of motA protein.') After 10 min on ice, transcription reactions were initiated by the addition Unlike the case with unmodifiedpolymerase, the 290-base transcriptisnotdetected with modified polymerase in the absenceof the motA protein (lanes 2 and 6 ) .These motA-dependent transcripts of asizepredictedfor initiationsaround position 1100 is observed. Addition, the strong promotefror unmodified RNA polymer- analysis of the RNA synthesized from Puvsinx vitro yields a ase at position 580 is still active using the RNA polymerase discrete band (Fig. 7A), demonstrating that thheeterogeneous from infected cells either with or without motA protein. This is notthe case at leastunderthese in vitro transcription conditions
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