scholarly journals The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

1991 ◽  
Vol 11 (7) ◽  
pp. 3515-3521 ◽  
Author(s):  
A Krauskopf ◽  
E Bengal ◽  
Y Aloni
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Cell Extract ◽  
Elongation Factors ◽  
Minute Virus Of Mice ◽  
Whole Cell ◽  
Minute Virus ◽  
Whole Cell Extract

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

scholarly journals The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors

1991 ◽  
Vol 11 (7) ◽  
pp. 3515-3521
Author(s):  
A Krauskopf ◽  
E Bengal ◽  
Y Aloni
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Cell Extract ◽  
Elongation Factors ◽  
Minute Virus Of Mice ◽  
Whole Cell ◽  
Minute Virus ◽  
Whole Cell Extract

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae

1991 ◽  
Vol 11 (9) ◽  
pp. 4555-4560 ◽  
Author(s):  
M Woontner ◽  
P A Wade ◽  
J Bonner ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Transcription System ◽  
Upstream Activation Sequence ◽  
Activation Sequence

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

scholarly journals FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation

2002 ◽  
Vol 22 (21) ◽  
pp. 7543-7552 ◽  
Author(s):  
Subhrangsu S. Mandal ◽  
Helen Cho ◽  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Danny Reinberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Carboxy Terminal Domain ◽  
Transcription Factor Iif ◽  
Transcript Elongation ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.

Structural insight into nucleosome transcription by RNA polymerase II with elongation factors

Science ◽  
2019 ◽  
Vol 363 (6428) ◽  
pp. 744-747 ◽  
Author(s):  
Haruhiko Ehara ◽  
Tomoya Kujirai ◽  
Yuka Fujino ◽  
Mikako Shirouzu ◽  
Hitoshi Kurumizaka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Chromosomal Dna ◽  
Elongation Factors ◽  
Cryo Electron Microscopy ◽  
Structural Insight ◽  
Insight Into

RNA polymerase II (RNAPII) transcribes chromosomal DNA that contains multiple nucleosomes. The nucleosome forms transcriptional barriers, and nucleosomal transcription requires several additional factors in vivo. We demonstrate that the transcription elongation factors Elf1 and Spt4/5 cooperatively lower the barriers and increase the RNAPII processivity in the nucleosome. The cryo–electron microscopy structures of the nucleosome-transcribing RNAPII elongation complexes (ECs) reveal that Elf1 and Spt4/5 reshape the EC downstream edge and intervene between RNAPII and the nucleosome. They facilitate RNAPII progression through superhelical location SHL(–1) by adjusting the nucleosome in favor of the forward progression. They suppress pausing at SHL(–5) by preventing the stable RNAPII-nucleosome interaction. Thus, the EC overcomes the nucleosomal barriers while providing a platform for various chromatin functions.

scholarly journals Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

2009 ◽  
Vol 106 (17) ◽  
pp. 6956-6961 ◽  
Author(s):  
Karen Zhou ◽  
Wei Hung William Kuo ◽  
Jeffrey Fillingham ◽  
Jack F. Greenblatt
Keyword(s):  
Histone Modification ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Consensus Sequence ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Histone H2b ◽  
Paf Complex

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

scholarly journals Host Cell Specificity of Minute Virus of Mice in the Developing Mouse Embryo

2004 ◽  
Vol 78 (17) ◽  
pp. 9474-9486 ◽  
Author(s):  
Refael Itah ◽  
Jacov Tal ◽  
Claytus Davis
Keyword(s):  
Host Cell ◽  
Mouse Embryo ◽  
Cell Types ◽  
Late Gestation ◽  
Productive Infection ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Overlapping Sets

ABSTRACT Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.

scholarly journals Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein–DNA interactions

1997 ◽  
Vol 69 (2) ◽  
pp. 101-110 ◽  
Author(s):  
ALAN TAYLOR ◽  
LIQUN ZHANG ◽  
JOHN HERRMANN ◽  
BEI WU ◽  
LARRY KEDES ◽  
...  
Keyword(s):  
Protein Binding ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Specific Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Nuclear Extracts

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to regions 5′ and 3′ of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3′ end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

scholarly journals Novel PKCη Is Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

2003 ◽  
Vol 77 (14) ◽  
pp. 8048-8060 ◽  
Author(s):  
Sylvie Lachmann ◽  
Jean Rommeleare ◽  
Jürg P. F. Nüesch
Keyword(s):  
Protein Kinase ◽  
Nonstructural Protein ◽  
Brdu Incorporation ◽  
Dominant Negative Mutant ◽  
Rolling Circle Replication ◽  
Minute Virus Of Mice ◽  
Rolling Circle ◽  
Minute Virus

ABSTRACT The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKCλ) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCη phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKCλ for rolling circle replication. Moreover, this role of PKCη was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCη mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically 32P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCηDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCη in the nuclear periphery, suggesting that besides being a target for PKCη, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle.

scholarly journals Regulation of Minute Virus of Mice NS1 Replicative Functions by Atypical PKCλ In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 433-442 ◽  
Author(s):  
Jürg P. F. Nüesch ◽  
Sylvie Lachmann ◽  
Romuald Corbau ◽  
Jean Rommelaere
Keyword(s):  
Dna Replication ◽  
Dominant Negative ◽  
Progeny Virus ◽  
Ns1 Protein ◽  
Viral Dna ◽  
Minute Virus Of Mice ◽  
Viral Dna Replication ◽  
Minute Virus

ABSTRACT Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKCλ mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKCλ. Our data clearly demonstrate that NS1 is a target for PKCλ phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKCλ which, unlike PKCζ, accumulates in the nuclear compartment of infected cells.

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