Nascent RNA cleavage by purified ternary complexes of vaccinia RNA polymerase.
Ternary complexes of vaccinia virus RNA polymerase containing 3'-OMeGMP-arrested transcripts were purified by native gel electrophoresis. These complexes resumed elongation in situ when gel slices were incubated with magnesium and NTPs. Elongation occurred in the absence of pyrophosphate, suggesting that the blocking 3'-OMeGMP residue was removed via a novel pathway. We show that purified elongation complexes contain an intrinsic nuclease activity that shortens nascent RNA from the 3'-end. RNA cleavage was absolutely dependent on a divalent cation and was stimulated by CTP. The initial 5' cleavage product remained associated with the ternary complex and could be elongated in the presence of NTPs. Multiple stepwise cleavages generated progressively shorter chains. Purified ternary complexes containing 3'-OH-terminated RNAs also displayed nuclease activity. Involvement of the vaccinia RNA polymerase subunit rpo30 in the transcript-shortening reaction is suggested based on sequence similarity of rpo30 to mammalian protein SII (TFIIS), an extrinsic transcription factor required for nascent RNA cleavage by RNA polymerase II (Reines, D. (1991) J. Biol. Chem. 267, 3795-3800).
Hagler
J
J
Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.
Shuman
S
S
D001483:Base Sequence; D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology; D004277:DNA, Single-Stranded; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D012313:RNA / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology*
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D009711:Nucleotides; D012315:RNA Caps; D003035:Cobalt; D008345:Manganese; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium