Abstract
At late times after infection with SV40, a prematurely terminated transcript that initiates at the major late promoter (MLP) and has a 3'-end about 95 nucleotides downstream has been identified and termed an attenuated RNA (Hay, N., Skolnik-David, H., and Aloni, Y. (1982) Cell 29, 183-193). The DNA template of the attenuated RNA has two regions of dyad symmetry, and the attenuated RNA can therefore fold into two hairpin elements. The hairpin element at the 3'-end of the attenuated RNA is followed by a stretch of Us and resembles a rho-independent terminator in prokaryotes. We have suggested that folding of the RNA into two hairpin elements will lead to a block of transcription elongation. Using site-directed mutagenesis, we created two templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared between the templates in a cell-free extract. We have shown that RNA polymerase II recognizes the SV40 sequence that leads to a block of transcription elongation, even when it is under the control of the MLP of adenovirus 2. The extent of the block of transcription elongation is directly dependent on the stability of the hairpin structure of the RNA as assessed by a comparison of transcription of the wild-type and mutated templates. The addition of Sarkosyl and transcription at an elevated temperature during the elongation reaction enhanced the production of the attenuated RNA from all templates.
Highlights
At late times after infection with SV40,a prema- Chen-Kiang, 1984; Mok et al, 1984; Seiberg et al, 19871, turely terminated transcript that initiateast themajor transcription from the P4 promoter of minute virus of mice late promoter(MLP) and hasa 3’-end about 95 nucle- (Ben-Asher andAloni, 1984; Resnekov and Aloni, 1989), and otides downstream has been identified and termedan transcription from thelong terminal repeat of human immuattenuated RNA (Hay, N., gkolnik-David, H., and nodeficiency virus, type 1 (Kao et al, 1987)
Time Course ut 37.5 "C of theThree Constructs-While described an inuitro block of transcription elongation located analyzing the kinetics of the transcription reactions on one about 95 nucleotides downstream from the MLP of SV40 of theconstructs at different temperatures (Fig. 4B), we (Hay etuL,1982)
We have shown that the RNA polymerase noticed the appearance of a transcript of about 155nucleotides I1 recognizes the SV40 sequence that which was sensitive to 0.5 pg/ml a-amanitin
Summary
Wild-type and Mutated Secondary Structures-We chose to examine the effect of the putative SV40 secondary structure upstream of the stretch of Us on transcription elongation by using a cell-free system. For this analysis, we cloned the SV40 sequences downstream of the MLP ofAd2. The(3 4) RNA produced with the addition of Sarkosyl to the transcriptiorenaction is about 5nucleotides longer than that produced without Sarkosyl This may indicate that Sarkosyl causes transcription to halt farther down within the stretch of Us
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
