Effect of the 21-bp repeat upstream element on in vitro transcription from the early and late SV40 promoters (original) (raw)
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Molecular and Cellular Biology, 1985
The presence of a surrogate TATA box sequence located ca. 30 nucleotides upstream of the major late RNA start site at nucleotide position (np) 325 (Brady et al., Cell 31:625-633, 1982) has been confirmed, and its structural specificity has been determined by the generation of additional base substitution mutations at the KpnI restriction site (np 294) in cloned simian virus 40 DNA. Two mutants generated new RNA initiation sites upstream of the np 325 start site and continued to utilize the authentic start site, but with decreased efficiency. The replacement of either one or both cytosines by thymines at np 298 and np 299 specifically enhanced in vitro transcription from the np 325 start site by 430 and 800%, respectively. This enhancement was due to conversion of the simian virus 40 late promoter present in the wild-type virus to a sequence that is similar to the TATA box present in the simian virus 40 early promoter.
Journal of virology, 1993
The simian virus 40 large T antigen is a promiscuous transcriptional activator of many viral and cellular promoters. We show that the promoter structure necessary for T antigen-mediated transcriptional activation is very simple. A TATA or initiator element is required, in addition to an upstream factor-binding site, which can be quite variable. We found that promoters containing an SP1-, ATF-, AP1-, or TEF-I-binding site, in conjunction with a TATA element, can all be activated in the presence of T antigen. In addition, preference for specific TATA elements was indicated. Promoters containing the HSP70 TATA element functioned better than those with the adenovirus E2 TATA element, while promoters containing the simian virus 40 (SV40) early TATA element failed to be activated. In addition, simple promoters containing the initiator element from the terminal deoxynucleotidyltransferase gene could be activated by T antigen. The SV40 late promoter, a primary target for T antigen transcrip...
Nucleic Acids Research, 1983
We have constructed deletion and point mutations within the Simian virus 40 (SV40) early promoter region which contains two tandemly repeated 21 bp sequences and a related 22 bp sequence (the "upstream" 21 bp repeat region). After transfection into permissive CV-1 cells and non-permissive mouse 3T3-4E cells, the effect of the mutations on early gene expression was studied by measuring T-antigen production, using indirect immunofluoresence. Our results demonstrate that the 21 bp repeat region, and in particular the six GC-rich motifs 5'-CCGCCC-3' which are repeated in this region constitute an important element of the SV40 early promoter. Surprisingly, we found that the requirement for the 21 bp repeat region for early gene expression was partially fulfilled even when it was in the inverted orientation.
Journal of virology, 1991
Simian virus 40 (SV40) large T antigen is a promiscuous transcriptional activator of many viral and cellular promoters. The SV40 late promoter, a primary target for T-antigen transcriptional activation, contains a previously described T-antigen-activatable binding site (SV40 nucleotides 186 to 225). The T-antigen-activatable binding site element contains overlapping octamer (Oct)- and SPH (TEF-1)-binding sites (Oct/SPH site). Using this Oct/SPH site as an upstream element in a simple promoter, we show that the SPH sites are necessary for transcriptional activation by T antigen. In addition, we show that when Oct 1 is overproduced, it can eliminate T-antigen-mediated transcriptional activation, as well as basal activity, from the simple Oct/SPH promoter as well as the intact SV40 late promoter. This suggests that one function of T antigen in transcriptional activation of the late promoter is to alter factor binding at the Oct/SPH region to favor binding of factors to the SPH sites.
Nucleic Acids Research, 1984
Using quantitative S1 nuclease analysis and recombinants which contain the SV40 early promoter region linked to the rabbit p-globin gene coding sequence, we have studied the effect of deletion, inversion and point mutations, located within the 21 bp repeat region, on initiation of transcription from the early-early and late-early startsites in the absence of T-antigen. Our data establish unequivocally that the six GC-rich repeats present in the 21 bp repeat region are essential elements of both the early-early and lateearly promoters and that they are not redundant, since mutations, which affect only the GC-rich repeat most proximal to the TATA box, decrease drastically the activity of the early-early promoter, but increase that of the late-early promoter. On the other hand, the four GC-rich repeats most proximal to the 72 bp repeat are common elements of the two overlapping earlyearly and late-early promoters. Our results, which confirm that the earlyearly promoter is stronger than the late-early one, also support our previous suggestion that they are in competition for the transcriptional machinery. The general organization of the SV40 early promoter region is discussed.
Cell, 1983
Activation of gene expression by the SV40 72 bp repeat was studied at the transcriptional level by quantitative S1 nuclease mapping of total RNA isolated from Hela cells transfected with chimeric conalbumin promoter-SV40 early gene recombinants. Our results demonstrate that, irrespective of its orientation, the 72 bp repeat is a potentiator of initiation of transcription from "TATA"-box-dependent and -independent "natural" or "substitute" promoter elements. In addition, we show that potential proximal promoter sequences are activated in preference to more distal ones. These results are consistent with the bidirectional entry site model for transcription activation by the 72 bp repeat.
Journal of Virology
The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatog...
Stereoalignment requirements for activation of transcription by the Simian virus 40 enhancer
Nucleic Acids Research, 1990
We investigated the contribution to the enhancer activity of protein-protein interactions between specific trans-acting factors bound to neighbouring sequence elements of the SV40 early enhancer-promoter region. To this end, we altered the distance between the SV40 enhancer-promoter elements by inserting systematically increasing lengths of spacer DNA. We show here that the level of transcription from the SV40 early promoter decreases with the spacing between enhancer and promoter regions. In the case of insertions shorter than 125 bp, the promoter activity exhibits a strong dependence on insertion lengths of multiples of about 10 bp. This periodic effect is no longer observed for layer insertions, reflecting the torsional flexibility of DNA. These data provide evidence that, between promoter and/or enhancer elements, periodic interactions of transcriptional nucleoprotein complexes exist and stereospecific alignments are necessary to obtain an efficient initiation of transcription from the SV40 early promoter.
Proceedings of the National Academy of Sciences, 1985
Transcriptional control signals required for tumor (T)-antigen trans-activation ofthe simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the transacting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72-bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important.
Simian virus 40 tandem repeated sequences as an element of the early promoter
Proceedings of the National Academy of Sciences, 1981
On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).