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

Abstract

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 alpha or beta position. 5'-terminal cap structures (7mGpgamma pbeta palpha X) on newly synthesized RNA were analyzed by digestion with nuclease P1 or with ribonuclease T2/bacterial alkaline phosphatase. Cap structures obtained after labeling with alpha-32P-GTP show that the 32P is found only adjacent to the 7mG residue (i.e., in the gamma position) and adjacent to the penultimate Gm or G nucleotide (i.e., in the alpha position). Analysis of RNA synthesized in the presence of beta-32P-ATP, however, shows GpppA cap structures which are labeled only in the beta position. In the presence of beta-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 wild-types SV40 the distribution of virus-specific caps is similar when labeled either with beta-32P-ATP or with alpha-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.

Nuclear Protein Interactions with the Human KDR/flk-1 Promoter in Vivo

“…To explore this possibility, we performed in vivo footprinting of the proximal promoter of KDR/flk-1 by LM-PCR in cells treated with DNase I after permeabilization with lysolecithin. This permeabilization technique does not inhibit transcription initiation and should therefore maintain a faithful promoter architecture for probing with DNase I (27). Compared with in vitro DNase I-treated purified genomic DNA, HUVEC genomic DNA treated in vivo was strongly protected over the four Sp1 sites defined as being important in our previous in vitro experiments (Fig.…”

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…Notably, this approach is different from the two previous ones as it leaves the rest of the RNA body completely unlabeled. It is also possible to obtain RNA selectively radiolabeled at the b-phosphate of the cap by adding an appropriate [b-32 P] NTP to the transcription reaction [42]. However, to our knowledge, this approach is rarely used, likely owing to a lack of commercial availability of [b-32 P] NTPs.…”

“…The identity of the cap structures can be confirmed by isolation and further digestion by a pyrophosphatase with broad specificity, such as tobacco acid pyrophosphatase (TAP), which cleaves both triphosphate bonds. This approach has been used to identify 5 0 ends of eukaryotic and viral mRNAs, snRNAs, and snoRNAs [42][43][44][45], as well as to verify the P-labeled RNAs using in vitro transcription followed by enzymatic capping. Depending on the template sequence and 'hot' NTP used in the transcription reaction, RNA can be labeled at the a position within the cap triphosphate bridge (1) or at the first phosphodiester bond of transcribed RNA (2).…”

Synthesis of Prematurely Terminated Late Transcripts of Polyoma Virus DNA is Resistant to Inhibition by 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole

“…However, there is some indirect evidence that capping may happen elsewhere than at the initial nucleotide in an RNA chain (Schibler & Perry, 1976;Spencer et al, 1978). On the other hand, recent studies using permeabilized cells or isolated nuclei (Contreras & Fiers, 1981;Gidoni et al, 1981) and in vitro transcription systems (Handa et al, 1981) have shown that initiation of transcription can take place at capping sites on the DNA of the related simian virus 40 (SV40). The present study was undertaken to provide independent evidence for the in vivo location of transcriptional initiation sites.…”