High specific activity [beta-32P]ATP and [beta-32P]CTP were used to study in vitro transcriptional initiation and subsequent capping of simian virus 40 (SV40) early and later RNAs. More than 40% of the capped SV40 RNA synthesized in vitro was also polyadenylylated. With [beta-32P]ATP, only adenosine-containing caps were labeled and the incorporated radioactive phosphate was found exclusively in the beta position. Cap digestion patterns showed extensive qualitative and quantitative similarities between these 32P-labeled caps and caps labeled in vivo [Canaani, D., Kahana, C., Mukamel, A. & Groner, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 3078--3082]. With [beta-32P]CTP, only early SV40 RNA was labeled, consistent with the absence of cytosine-containing caps in late transcripts. The [beta-32P]CTP-labeled cap was identified as m7GpppCmpU, which was previously identified as the major cap of in vivo labeled early SV40 mRNA [Kahana, C., Gidoni, D., Canaani, D. & Groner, Y. (1981) J. Virol. 37, 7--16]. This experiment provides biochemical evidence for eukaryotic RNA polymerase II initiation of transcription with CTP. The data imply that, on SV40 DNA, RNA polymerase II initiates transcription at multiple nucleotide sequences and capping occurs at the initiator nucleotide.
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