Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit

1984 ◽  
Vol 4 (10) ◽  
pp. 2031-2040
Author(s):  
M Mok ◽  
A Maderious ◽  
S Chen-Kiang
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Premature Termination ◽  
Adenovirus Type ◽  
Transcriptional Unit ◽  
Late Gene ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Adenovirus Type 5

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

scholarly journals Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

1984 ◽  
Vol 4 (10) ◽  
pp. 2031-2040 ◽  
Author(s):  
M Mok ◽  
A Maderious ◽  
S Chen-Kiang
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Premature Termination ◽  
Adenovirus Type ◽  
Transcriptional Unit ◽  
Late Gene ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Adenovirus Type 5

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

scholarly journals Unusual Properties of Adenovirus E2E Transcription by RNA Polymerase III

2003 ◽  
Vol 77 (7) ◽  
pp. 4015-4024 ◽  
Author(s):  
Wenlin Huang ◽  
S. J. Flint
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Transcription Unit ◽  
Promoter Sequences ◽  
Polymerase Iii ◽  
Infected Cells ◽  
Adenovirus Type 5 ◽  
Rna Polymerase Ii Transcription

ABSTRACT In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5′ end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T1 protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit

1990 ◽  
Vol 10 (10) ◽  
pp. 5562-5564
Author(s):  
S Buratowski ◽  
P A Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Adenovirus Type ◽  
Late Promoter ◽  
Terminal Domain ◽  
Late Transcription ◽  
Major Late Promoter

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

scholarly journals Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

1982 ◽  
Vol 2 (10) ◽  
pp. 1295-1298 ◽  
Author(s):  
B F Cheetham ◽  
D C Shaw ◽  
A J Bellett
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Ornithine Decarboxylase ◽  
Adenovirus Type ◽  
S Phase ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Cellular Dna ◽  
Adenovirus Type 5

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.

scholarly journals CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

2021 ◽  
Author(s):  
Michael Tellier ◽  
Justyna Zaborowska ◽  
Jonathan Neve ◽  
Takayuki Nojima ◽  
Svenja Hester ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Premature Termination ◽  
Transcription Termination ◽  
Elongation Factor ◽  
Protein Coding ◽  
Pol Ii ◽  
Working Together ◽  
Rna Maturation ◽  
Nascent Transcripts

CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allows the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an ATP analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

scholarly journals RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures

1991 ◽  
Vol 11 (3) ◽  
pp. 1508-1522
Author(s):  
S C Linn ◽  
D S Luse
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Adenovirus Type ◽  
Dnase I ◽  
Transcription Complexes ◽  
Adenovirus Type 2

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.

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