Vaccinia virus has a genome of about 85,000 base pairs that encodes approximately 200 polypeptides. These genes are expressed are expressed within the cytoplasm in a coordinated fashion so that some polypeptides are made before and others after DNA genes are packaged within the infectious particle while those needed for late transcription are present in the cytoplasm of infected cells.
The aim of this project is to determine the mechanisms regulating transcription. This involves the enzymes and protein factors, the structure of the RNA and the DNA signals. Progress in understanding early transcription has been made this past year. Three components have been purified from virus particles; a multi subunit DNA-dependent RNA polymerase; a transcription initiation factor that binds specifically to early promoters and has an associated DNA-dependent ATPase; and a transcription termination factor that is a component of the capping enzyme complex. The termination signal, originally identified as TTTTTNT was shown to be recognized after transcription and hence is UUUUUNU. The poly (A) polymerase from vaccinia virions was further characterized with regard to its specificity for nucleotides. The type I DNA topoisomerase from vaccinia virions was purified, the sequence of the NH2-terminus determined, the gene mapped, and the enzyme expressed in high yield in E. coli. The 5' ends of late mRNAs were shown to consist of capped poly (A) leaders of variable length. A transcription system that specifically transcribes late genes was developed from extracts of infected cells and a transcription factor was partially purified. The in vitro system synthesizes late mRNA with the 5'poly(A)leader.
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