Stalling by RNA polymerase II in the polyomavirus intergenic region is dependent on functional large T antigen

Bertin, John; Sunstrom, Noelle-Ann; Jain, Poonam; Acheson, Nicholas H.

doi:10.1016/0042-6822(92)90594-f

Cited by 9 publications

(17 citation statements)

References 54 publications

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“…No such RNA has been previously been reported in the case of polyomavirus. What has been demonstrated, however, is that RNA polymerase II stalls at many sites on both the early and the late strands (8,9,11,26,41,42), especially in regions that contain a large-T-antigen-binding site. Thus, this RNA may be a result of stalling polymerases.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Chen

Fluck

2001

J Virol

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Kinetic studies of the accumulation of early and late transcripts, early and late proteins, genomes, and live virus, during the lytic cycle of murine polyomavirus wild-type A2, were carried out in synchronized NIH 3T3 cells released from G 0 by the addition of serum after infection. This first-time simultaneous analysis of all parameters of the virus life cycle led to new insights concerning the transcriptional control at the early-to-late transition. During the early phase, early transcripts were synthesized at very low levels, detectable only by reverse transcription-PCR, from 6 h postinfection (hpi). Large T protein could be detected by 8 hpi (while infected cells were in the G 1 phase). The level of expression of the middle T and small T proteins was lower than that of large T at all times, due, at least in part, to a splicing preference for the large-T 5 splice site at nucleotide 411. A large increase in the level of both early and late transcripts coincided closely with the detection in mid-S phase of viral genome amplification. Thereafter, both classes of transcripts continued to further accumulate up to the end of the experiments (48 hpi). In addition, during the late phase, "giant" multigenomic transcripts were synthesized from the early as well as the late promoter. Thus, a major type of transcriptional control appears to be applied similarly to the transcription of both early and late genes. This view differs from that in the literature, which highlights the enhancement of late transcription and the repression of early transcription. However, despite this parallel transcriptional control, additional regulations are applied which result in higher levels of late compared to early transcripts, as previously described. In the accompanying article, a key role for middle T and/or small T in this late-phase enhancement of early and late transcription is demonstrated (16). Other novel findings, e.g., the synthesis of a very abundant short early promoter proximal RNA, are also described.Polyomaviruses have served as important model systems for the understanding of various aspects of cellular organization and function, such as chromatin structure, the role of enhancers in gene expression, splicing mechanism, and the composition of the DNA replication machinery, to name a few. This is in part a consequence of the DNA nature and small size of the genomes of this virus family, which render viral replication highly dependent on, and hence compatible with, cellular processes. As a result, and with the added interest in their capacity to induce neoplastic transformation, these viruses have been the subjects of extensive studies.Surprisingly at this late stage of analysis, there has been no comprehensive study of the temporal relationships between all the various steps of the lytic cycle. Current references to the timing of these steps invariably quote the 1981 review by Acheson (2), which offers a thorough integration of the studies available at the time. However, these studies were done at different times, in differ...

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Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Chen

Fluck

2001

J Virol

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“…It is possible that this is due to steric hindrance or stalling of bound RNA and DNA polymerases. Stalling of RNA polymerase II can occur in vivo during transcription due to DNA lesions or nucleotide-specific binding of proteins (Sunstrom et al, 1992). Likewise, DNA polymerase has been shown to stall during DNA amplification due to stable secondary structures from base repeats (Krasilnikova et al, 1998) or bulky DNA lesions (Yan et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Quantitative analysis of EDC‐condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

Mann

McKnight

Melechko

et al. 2007

Biotech & Bioengineering

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Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. Polymerase chain reaction (PCR) and in vitro transcription (IVT) were used to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. This investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

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“…Cells and viruses. Growth of mouse 3T6 and primary baby mouse kidney cells and production of virus stocks were carried out as described previously (9). The wild-type polyomavirus strain (AT3-Modori) used in this st'ddy contains four additional restriction sites which were introduced, by oligonucleotide-directed mutagenesis, into strain AT3 adjacent to large T-antigen-binding sites C, B, A, and 1/2 (63).…”

Section: Methodsmentioning

confidence: 99%

“…Sequence analysis of the various regions of termination has not revealed the existence of a common terminator element, and in only a few cases have specific elements involved in terminating transcription by RNA polymerase II been identified (3,12,13,30,60,66). RNA polymerase II has been observed to stall or terminate transcription within several viral and cellular transcription units in regions on the template DNA which contain binding sites for proteins (3,9,13,17,18,47,58,62). It has been proposed that DNA-binding proteins may function as stalling or termination signals which impede the progression of RNA polymerases through specific regions of a transcription unit.…”

mentioning

confidence: 99%

Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA

Bertin

Sunstrom

Acheson

1993

J Virol

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During transcription of the late strand of polyomavirus DNA, RNA polymerase II stalls and accumulates nearby the binding sites on viral DNA recognized by polyomavirus large T antigen. Stalling by RNA polymerases is eliminated when thermolabile large T antigen is inactivated by using a temperature-sensitive virus mutant (J. Bertin, N.-A. Sunstrom, P. Jain, and N. H. Acheson, Virology 189:715-724, 1992). To determine whether stalling by RNA polymerases is mediated through the interaction of large T antigen with one or more of its binding sites, viable polyomavirus mutants that contain altered large-T-antigen-binding sites were constructed. Point mutations were introduced by site-directed mutagenesis into the multiple, clustered G(A/G)GGC pentanucleotides known to be the target sequence for large T-antigen binding. Mutation of the G(A/G)GGC pentanucleotides in the first two binding sites encountered by RNA polymerases in the intergenic region (sites C and B) had no detectable effect on stalling as measured by transcriptional run-on analysis. However, mutation of the two GAGGC pentanucleotides in binding site A, which lies adjacent to the origin of viral DNA replication, eliminated stalling by RNA polymerases. We conclude that binding of large T antigen to site A blocks elongation by RNA polymerase II. Further characterization of virus containing mutated site A did not reveal any effects on early transcription levels or on virus DNA replication. However, the mutant virus gave rise to small plaques, suggesting impairment in some stage of virus growth. Stalling of RNA polymerases by large T antigen bound to the intergenic region of viral DNA may function to prevent transcription from displacing proteins whose binding is required for the normal growth of polyomavirus.

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Stalling by RNA polymerase II in the polyomavirus intergenic region is dependent on functional large T antigen

Cited by 9 publications

References 54 publications

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Quantitative analysis of EDC‐condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA

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