RNA polymerase-associated transcription specificity factor encoded by vaccinia virus.
Vaccinia virus encodes a multisubunit DNA-dependent RNA polymerase (EC 2.7.7.6) that is packaged in the infectious virus particle. This polymerase was found to contain a submolar polypeptide of approximately 85 kDa in addition to the core subunits, which consist of two larger and several smaller polypeptides. The polymerase containing the 85-kDa polypeptide was separated from the core polymerase by column chromatography. Although the core polymerase actively transcribed heterologous single-stranded DNA, only the form with the associated 85-kDa polypeptide could act in conjunction with an early stage-specific factor to transcribe double-stranded DNA containing a vaccinia virus early promoter. Peptide sequencing established that the RNA polymerase-associated 85-kDa protein was derived from the vaccinia virus H4L open reading frame, which encodes a 94-kDa polypeptide that we named RAP94. RAP94 is not closely related to prokaryotic sigma 70 or eukaryotic RAP30 RNA polymerase-binding proteins, although there are short regions of sequence similarity. The specific association of RAP94 with viral RNA polymerase was corroborated with antibody raised to a recombinant fusion protein. Unlike the previously defined subunits of vaccinia virus RNA polymerase, RAP94 is synthesized exclusively late in infection, and synthesis could be prevented by a DNA replication inhibitor. The role of RAP94 in mediating specific transcription was demonstrated by using an extract from cells in which the H4L open reading frame had been transiently expressed.
Ahn
B Y
BY
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Moss
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016679:Genome, Viral; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins
D007527:Isoenzymes; D014157:Transcription Factors; D014764:Viral Proteins; C064449:early transcription factor protein, Vaccinia virus; D012321:DNA-Directed RNA Polymerases