scholarly journals Rad26p, a transcription-coupled repair factor, is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner in vivo

2009 ◽  
Vol 38 (5) ◽  
pp. 1461-1477 ◽  
Author(s):  
Shivani Malik ◽  
Priyasri Chaurasia ◽  
Shweta Lahudkar ◽  
Geetha Durairaj ◽  
Abhijit Shukla ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dependent Manner ◽  
Dna Lesion ◽  
Repair Factor ◽  
Transcription Coupled Repair

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 P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner

2005 ◽  
Vol 16 (11) ◽  
pp. 5304-5315 ◽  
Author(s):  
Archa H. Fox ◽  
Charles S. Bond ◽  
Angus I. Lamond
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Similarity ◽  
Dependent Manner ◽  
Rna Pol Ii ◽  
Nuclear Processes ◽  
Rna Polymerase Ii Transcription

P54nrb is a protein implicated in multiple nuclear processes whose specific functions may correlate with its presence at different nuclear locations. Here we characterize paraspeckles, a subnuclear domain containing p54nrb and other RNA-binding proteins including PSP1, a protein with sequence similarity to p54nrb that acts as a marker for paraspeckles. We show that PSP1 interacts in vivo with a subset of the total cellular pool of p54nrb. We map the domain within PSP1 that is mediating this interaction and show it is required for the correct localization of PSP1 to paraspeckles. This interaction is necessary but not sufficient for paraspeckle targeting by PSP1, which also requires an RRM capable of RNA binding. Blocking the reinitiation of RNA Pol II transcription at the end of mitosis with DRB prevents paraspeckle formation, which recommences after removal of DRB, indicating that paraspeckle formation is dependent on RNA Polymerase II transcription. Thus paraspeckles are the sites where a subset of the total cellular pool of p54nrb is targeted in a RNA Polymerase II-dependent manner.

scholarly journals Transcriptional Cofactor CA150 Regulates RNA Polymerase II Elongation in a TATA-Box-Dependent Manner

1999 ◽  
Vol 19 (7) ◽  
pp. 4719-4728 ◽  
Author(s):  
Carlos Suñé ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Dependent Manner ◽  
Functional Requirements ◽  
Tat Protein ◽  
Transcription Complexes ◽  
Hiv 1

ABSTRACT Tat protein strongly activates transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by enhancing the elongation efficiency of RNA polymerase II complexes. Tat-mediated transcriptional activation requires cellular cofactors and specific cis-acting elements within the HIV-1 promoter, among them a functional TATA box. Here, we have investigated the mechanism by which one of these cofactors, termed CA150, regulates HIV-1 transcription in vivo. We present a series of functional assays that demonstrate that the regulation of the HIV-1 LTR by CA150 has the same functional requirements as the activation by Tat. We found that CA150 affects elongation of transcription complexes assembled on the HIV-1 promoter in a TATA-box-dependent manner. We discuss the data in terms of the involvement of CA150 in the regulation of Tat-activated HIV-1 gene expression. In addition, we also provide evidence suggesting a role for CA150 in the regulation of cellular transcriptional processes.

scholarly journals Rad26, the Transcription-Coupled Repair Factor in Yeast, Is Required for Removal of Stalled RNA Polymerase-II following UV Irradiation

PLoS ONE ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. e72090 ◽  
Author(s):  
Sounak Ghosh-Roy ◽  
Dhiman Das ◽  
Debarati Chowdhury ◽  
Michael J.Smerdon ◽  
Ronita Nag Chaudhuri
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Irradiation ◽  
Repair Factor ◽  
Transcription Coupled Repair

scholarly journals The FACT Complex Travels with Elongating RNA Polymerase II and Is Important for the Fidelity of Transcriptional Initiation In Vivo

2003 ◽  
Vol 23 (22) ◽  
pp. 8323-8333 ◽  
Author(s):  
Paul B. Mason ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Tata Binding Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Transcriptional Initiation ◽  
Pol Ii ◽  
Coding Regions

ABSTRACT The FACT complex facilitates transcription on chromatin templates in vitro, and it has been functionally linked to nucleosomes and putative RNA polymerase II (Pol II) elongation factors. In Saccharomyces cerevisiae cells, FACT specifically associates with active Pol II genes in a TFIIH-dependent manner and travels across the gene with elongating Pol II. Conditional inactivation of the FACT subunit Spt16 results in increased Pol II density, transcription, and TATA-binding protein (TBP) occupancy in the 3′ portion of certain coding regions, indicating that FACT suppresses inappropriate initiation from cryptic promoters within coding regions. Conversely, loss of Spt16 activity reduces the association of TBP, TFIIB, and Pol II with normal promoters. Thus, FACT is required for wild-type cells to restrict initiation to normal promoters, thereby ensuring that only appropriate mRNAs are synthesized. We suggest that FACT contributes to the fidelity of Pol II transcription by linking the processes of initiation and elongation.

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.

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