Purification and identification of a vaccinia virus-encoded intermediate stage promoter-specific transcription factor that has homology to eukaryotic transcription factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit.
Enzymes and factors, required for in vitro transcription of templates regulated by vaccinia virus intermediate stage promoters, are present in HeLa cells infected with vaccinia virus in the presence of an inhibitor of DNA replication. Previous studies indicated that in vitro transcription could be reconstituted by adding a partially purified transcription factor to the viral RNA polymerase and capping enzyme. By using an independent purification procedure, we isolated two vaccinia virus intermediate were necessary for transcription of several different intermediate stage promoter templates but not for early or late stage promoter templates. VITF-1 was purified to homogeneity, and the sequences of two tryptic peptides were mapped to the fourth open reading frame within the HindIII E fragment (E4L) of the vaccinia virus genome, which had previously been shown to encode an RNA polymerase subunit of 30 kDa (RPO30) with homology to eukaryotic transcription elongation factor SII. Co-chromatography of VITF-1 with the E4L-derived protein was demonstrated using specific antiserum. In addition, transcriptionally active recombinant VITF-1 was made by expressing the E4L open reading frame in Escherichia coli. Thus, E4L encodes a multifunctional protein, serving as a RNA polymerase subunit and a stage-specific transcription factor. The stepwise binding of capping enzyme, VITF-1, and VITF-2 to a DNA/viral RNA polymerase complex was demonstrated.
Rosales
R
R
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Harris
N
N
Ahn
B Y
BY
Moss
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002853:Chromatography, Liquid; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification*; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
C486562:CAPRIN1 protein, human; D018797:Cell Cycle Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; D014764:Viral Proteins; C018648:transcription factor S-II; D012321:DNA-Directed RNA Polymerases