Nucleic Acids Research, 1981, Vol. 9, No. 2 215-236
© 1981
MOLECULAR BIOLOGY |
Initiation of transcription by RNA polymerase II in permeable, SV40-infected or noninfected, CV1 cells; evidence for multiple promoters of SV40 late transcription
Laboratory of Molecular Biology, State University of Ghent Ledeganckstraat 35, B-9000 Ghent, Belgium
Received November 27, 1980.
CV1 cells were made permeable by treatment with lysolecithin and incubated in a transcription mixture containing ribonucleoside triphosphates including ATP or GTP 32P-labeled either in the 
or ß position. 5'-terminal cap structures ( 7m 
ß 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 -32P-GTP show that the 32P is found only adjacent to the 7mG residue ( i.e., in the position) and adjacent to the penultimate Gm or G nucleotide ( i.e., in the position). Analysis of RNA synthesized in the presence of ß-32P-ATP, however, shows GpppA cap structures which are labeled only in the ß position. In the presence of ß-32P-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 ß-32P-ATP or with -32P-GTP or with 32P-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.
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