Initiation of transcription by RNA polymerase II in permeable, SV40-infected or noninfected, CV1 cells; evidence for multiple promoters of SV40 late transcription

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Abstract
CV1 cells were made permeable by treatment with lysolecithin and incubated in a transcription mixture containing ribonucleoside triphosphates including ATP or GTP 32 P-labeled either in the α or β position. 5′-terminal cap structures ( 7m γ β α GpppX) on newly synthesized RNA were analyzed by digestion with nuclease P1 or with ribonuclease T2/bacterial alkaline phosphatase. Cap structures obtained after labeling with α- 32 P-GTP show that the 32 P is found only adjacent to the 7m G residue ( i.e., in the γ position) and adjacent to the penultimate Gm or G nucleotide ( i.e., in the α position). Analysis of RNA synthesized in the presence of β- 32 P-ATP, however, shows GpppA cap structures which are labeled only in the β position. In the presence of β- 32 P-GTP, only GpppG structures are labeled; these findings exclude the hypothesis that caps are synthesized from GTP and a monophosphate 5′-terminal RNA molecule. The results imply that the initial transcripts are used for cap formation, which indicates that the large majority ( if not all ) of capping sites correspond to initiation sites for transcription. In cells infected with wildtype SV40 the distribution of virus-specific caps is similar when labeled either with β- 32 P-ATP or with α- 32 P-GTP or with 32 P-phosphate. Thus, evidence is presented that heterogeneity of the cap structures in late SV40 is a consequence of independent initiation events and not of processing of a primary transcript followed by capping of the 5′ ends generated.