Binding of an analog of the simian virus 40 T antigen to wild-type and mutant viral replication origins

Tenen, Daniel G.; Haines, Lora L.; Livingston, David M.

doi:10.1016/0022-2836(82)90472-7

Cited by 33 publications

(32 citation statements)

References 27 publications

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“…3). Thus, pS3X lacks SV40 ori function but retains the early-promoter and Tantigen binding sites (16,58). To remove the normal earlypromoter region, pSp DNA was digested with PvuII and HindIlI endonucleases, and the large 4,342-bp fragment was purified by gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

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Complex regulation of simian virus 40 early-region transcription from different overlapping promoters.

Buchman¹,

Fromm²,

Berg³

1984

Mol. Cell. Biol.

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During simian virus 40 lytic infection there is a shift in initiation sites used to transcribe the early region, which encodes large T and small t antigens. Early in infection, transcription is initiated almost exclusively from sites that are downstream of the origin of DNA replication, whereas transcripts produced later are initiated mainly from sites on the upstream side. We have used mutant virus and specially constructed plasmid DNAs to investigate the factors regulating this transcriptional shift. In our studies simian virus 40 large T antigen appears to mediate the shift in transcription in two ways: first, T antigen represses transcription at the downstream sites late in infection by binding to the region where these RNAs are initiated; second, T antigen promotes transcription from sites on the upstream side by its ability to initiate replication or amplification, or both, of the template DNA. In addition, transcription from the downstream sites is heavily dependent on enhancer sequences located in the 72-base-pair repeat region, whereas transcription from the upstream sites late in infection does not require enhancer sequences. Thus, different overlapping promoters regulate simian virus 40 early-region expression in a manner that apparently coordinates the production of large T antigen with the increase in viral DNA.At present, the molecular mechanisms controlling gene expression in higher eucaryotes are only poorly understood. One of the best characterized systems which has been used to approach this problem is the early region of simian virus 40 (SV40) (1, 24, 56). The early region codes for two known proteins, large T antigen and small t antigen. Although the function of small t antigen remains obscure, large T antigen is known to be required for the initiation of viral DNA synthesis. These proteins are translated from differentially spliced RNAs which are transcribed, using the same promoter and polyadenylation signals. The SV40 early-region promoter is contained within a set of repeated segments adjacent to the origin of viral DNA replication (ori) (Fig. 1). Extensive analysis of this region has demonstrated the presence of three separate elements involved in SV40 early transcription (5, 16). At least one copy of a set of six short guanine-cytosine-rich repeats is necessary for early transcription. In addition, a sequence within the 72-base-pair (bp) repeat has been shown to be an essential element. Finally, a Goldberg-Hogness or TATA box sequence (19; M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978), imbedded within a larger 17-bp adenine-thymine (AT)-rich block, is responsible for positioning the 5' ends of early RNA.The SV40 early region is also one of the best characterized examples of regulation mediated by a defined protein. Large T antigen regulates its own synthesis by controlling the level of early-region transcription (33, 53, 57). Furthermore, T antigen binds to SV40 DNA at three adjacent sites overlapping ori and the early promoter (Fig. 1) that T-antigen binding a...

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Section: Methodsmentioning

confidence: 99%

“…1). In vitro, site I has the highest affinity for T antigen and site III has the lowest (58,60). Studies with deletion and point mutations suggest that T-antigen binding at sites I and II is involved in the initiation of DNA replication at ori (37,50,55).…”

mentioning

confidence: 99%

Complex regulation of simian virus 40 early-region transcription from different overlapping promoters.

Buchman¹,

Fromm²,

Berg³

1984

Mol. Cell. Biol.

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“…Binding of SV40 T-antigen to the viral DNA occurs at three adjacent sites (8,56,57), located 200 to 400 bp upstream of the SV40 major late RNA initiation site. Mutant templates which contain deletions in the T-antigen-binding sites were analyzed after being used to transfect CV-1 and COS-1 cells (Fig.…”

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confidence: 99%

trans Activation of the simian virus 40 late transcription unit by T-antigen.

Brady¹,

Khoury²

1985

Mol. Cell. Biol.

115

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We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and SI nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site H decreased T-antigen-mediated late gene expression approximately 10-to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.Analysis of the genes transcribed by RNA polymerase II has revealed a complex and interrelated set of promoter elements. Most eucaryotic genes transcribed by RNA polymerase II share a common upstream sequence about 25 nucleotides before the cap site (position -25). The GoldbergHogness (or TATA) box appears to function in vitro and in vivo in positioning the site of initiation of RNA synthesis (3,16,53,59). Several additional upstream transcriptional regulatory sequences have been identified which directly influence the efficiency of RNA synthesis. One of these elements is a guanine-cytosine (GC)-rich motif containing a characteristic hexanucleotide. In simian virus 40 (SV40), this sequence (GGGCGG) is represented twice in each of the three 21-base-pair (bp) tandem repeats and has recently been shown by Dynan and Tjian to bind a transcriptional factor, SP-1 (9, 10), which appears to play a role in both early and late viral RNA synthesis (7,11,15,27,43). A similar GC-rich sequence is located upstream of the herpesvirus thymidine kinase RNA initiation site and in a cellular promoter (42,50).Another set of transcriptional control elements, commonly referred to as enhancers (2,3,24,30,31,39,40,44,...

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“…1) (30,31) at the late-transcription side of the origin; its functional role in III is not yet known (32). DNase protection experiments (8)(9)(10)12), DNase and dimethyl sulfate cleavage inhibition patterns ("footprints") (8,(12)(13)(14), and alkylation-interference experiments (15) have been used to explore the binding interactions of T antigen with respect to origin sequences in considerable detail. A model has been proposed in which monomer units (12) recognize the pentanucleotide 5' G(T)-A-G-G-C 3' in binding regions I and II and 5' G(T)-G-G-G-C 3' in binding region III ( Fig.…”

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confidence: 99%

“…[5][6][7]. Experiments to determine the mechanisms of regulation by T antigen have attacked successfully the problem of identifying the DNA sequences with which the protein interacts (8)(9)(10)(11)(12)(13)(14)(15) but less definitively the characterization of bound structures (16)(17)(18)(19).…”

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Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution.

Mastrangelo¹,

Hough²,

Wilson³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Large tumor (T) antigen and its bound multimeric states are positioned by scanning transmission electron microscopy (STEM) within a few base pairs at control sequences of the simian virus 40 DNA origin of replication region. Proximal and distal edge positions for each multimer group match the end positions of previously mapped fragments protected from DNase cleavage. Since chance correspondence is shown to be extremely unlikely, STEM mass measurements, obtained concurrently with STEM map positions, indicate that the DNase fragments arise from bound monomers, dimers, trimers, and tetramers in binding region II and monomers, dimers, and trimers in binding region I.

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Binding of an analog of the simian virus 40 T antigen to wild-type and mutant viral replication origins

Cited by 33 publications

References 27 publications

Complex regulation of simian virus 40 early-region transcription from different overlapping promoters.

Complex regulation of simian virus 40 early-region transcription from different overlapping promoters.

trans Activation of the simian virus 40 late transcription unit by T-antigen.

Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution.

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