DNA sequences which influence the selection and efficient utilisation of transcriptional start sites of a eukaryotic gene by RNA polymerase II in vitro and in vivo

1986 ◽  
Vol 6 (11) ◽  
pp. 937-944
Author(s):  
Balazs J. Kovacs ◽  
Peter H. W. Butterworth
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Tata Box ◽  
Flanking Sequence ◽  
Eukaryotic Gene ◽  
Transcriptional Start Sites

Experiments are described which probe the relationship between three sequence elements which make up the eukaryotic RNA polymerase II promoter. A cloned eukaryotic gene, from which the TATA-box and 400 base pairs of Y-flanking sequence has been deleted, is still transcriptionally active in vivo (following its transfection into cultured mammalian cells) and in vitro. Deletion has appropriately positioned a cluster of five TATA box-like sequences upstream from multiple potential cap sites. Which cap sites are actually used can be predicted from the DNA sequence of TATA box-like sequences and their spatial relationship with respect to possible transcriptional start sites, although there appears to be some difference in cap site utilisation in vitro and in vivo. Data suggest that deletion has also removed “upstream” sequences which affect promoter function.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery

2014 ◽  
Vol 95 (8) ◽  
pp. 1640-1646 ◽  
Author(s):  
Gerald Barry ◽  
Mariana Varela ◽  
Maxime Ratinier ◽  
Anne-Lie Blomström ◽  
Marco Caporale ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Antiviral Response ◽  
Cellular Protein ◽  
Schmallenberg Virus ◽  
Cellular Protein Synthesis

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways.

scholarly journals Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033 ◽  
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

scholarly journals Wwp2-Mediated Ubiquitination of the RNA Polymerase II Large Subunit in Mouse Embryonic Pluripotent Stem Cells

2007 ◽  
Vol 27 (15) ◽  
pp. 5296-5305 ◽  
Author(s):  
Hui Li ◽  
Zhihong Zhang ◽  
Beibei Wang ◽  
Junmei Zhang ◽  
Yingming Zhao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Terminal Domain ◽  
Ubiquitin E3 Ligase ◽  
Lysine Residues

ABSTRACT Ubiquitination and the degradation of the large subunit of RNA polymerase II, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions.

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

scholarly journals The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro

1978 ◽  
Vol 176 (3) ◽  
pp. 715-725 ◽  
Author(s):  
T J C Beebee
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Dna Sequences ◽  
Acid Synthesis ◽  
Rna Molecules ◽  
Liver Nuclei

1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 × 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.

scholarly journals Analysis of Gene Induction and Arrest Site Transcription in Yeast with Mutations in the Transcription Elongation Machinery

2001 ◽  
Vol 276 (15) ◽  
pp. 11531-11538 ◽  
Author(s):  
Megan Wind-Rotolo ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Mrna Accumulation ◽  
Mrna Metabolism ◽  
Transcript Elongation

In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands. Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles. There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthroughin vivo. Saccharomyces cerevisiaestrains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs. Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation. Both mutants and a combined double mutant were defective in induction ofGAL1andENA1. This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants. These data are consistent with mutants in the Elongator complex, which show slow inductive responses. When a potentin vitroarrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation. These data suggest that an alternative elongation surveillance mechanism existsin vivoto overcome arrest.

scholarly journals Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription

2007 ◽  
Vol 409 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Marilena Cojocaru ◽  
Célia Jeronimo ◽  
Diane Forget ◽  
Annie Bouchard ◽  
Dominique Bergeron ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Affinity Purification ◽  
Initiation Site ◽  
Transcriptional Initiation ◽  
Initiation Phase ◽  
Genomic Location

The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.

In vitro analysis of a transcription termination site for RNA polymerase II

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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