Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II.

Hawley, Diane K.; Roeder, R G

doi:10.1016/s0021-9258(17)39577-7

Cited by 304 publications

(37 citation statements)

References 26 publications

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“…The same Drosophila cell line used to construct the Pol II iDNA library was used to make nuclear run-on probes in the presence or absence of the detergent sarkosyl. Sarkosyl treatment allows transcriptionally engaged (both transcribing and paused) Pol II to transcribe (12), but it prevents further rounds of initiation (28). Labeled run-on RNAs were made and hybridized to a representative group of iDNA library clones and control genes represented on the Southern blot shown in Figure 2.…”

Section: Resultsmentioning

confidence: 99%

Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila

Law

Hirayoshi

O’Brien

et al. 1998

Nucleic Acids Research

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A new method is described for cloning DNA sequences occupied by a specific protein on chromatin in vivo . The approach uses UV cross-linking to couple proteins covalently to DNA and the resulting complexes are then purified under stringent conditions. Particular adducts are immunoprocipitated with antibody to the protein of interest. The resulting DNA (iDNA) is amplified by PCR, cloned and characterized. The model system used was RNA polymerase II (Pol II), whose density on particular DNAs under various conditions is well documented. Pol II can exist in several states on DNA. While Pol II can simply be bound to DNA, the bulk of DNA-associated Pol II is transcriptionally engaged in either the transcribing or paused states. Paused Pol IIs that have previously been characterized are found at promoters and have the distinctive property that their transcription in isolated nuclei is stimulated by sarkosyl or high salt. Here we isolate and sequence DNAs that cross-link to Pol II molecules. We identify by nuclear run-on assays those DNAs that have Pol II engaged in transcription. Twenty one percent of the iDNA clones that have detectable transcriptionally engaged Pol II appear to be paused, in that they display sarkosyl-stimulated trancription in a nuclear run-on transcription assay. At least some of these map to the 5'-ends of genes. These results suggest that transcriptional pausing of Pol II is a general phenomenon in vivo.

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Section: Resultsmentioning

confidence: 99%

Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila

Law

Hirayoshi

O’Brien

et al. 1998

Nucleic Acids Research

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“…Single-round transcription was confirmed based upon quantitation of total 32 P signal (i.e. encompassing all detected transcripts ~20 to 216 nt in length) over time and further verified using experiments with added sarcosyl (0.2%), which prevents re-initiation (Hawley and Roeder, 1985;Hawley and Roeder, 1987). Transcription was stopped by addition of 150 µL of stop buffer (20 mM EDTA, 200 mM NaCl, 1% SDS).…”

Section: Methods Detailsmentioning

confidence: 99%

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

et al. 2019

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TFIIH, Mediator, and RNAPII (Figure S1). Experiments were completed with the native human HSP70 promoter (HSPA1B gene), because others have shown that it is a quintessential model for promoter-proximal RNAPII pausing (Core et al., 2012). Because chromatin per se does not appear to be an essential regulator of RNAPII pausing in Drosophila or mammalian cells Lai and Pugh, 2017;Li et al., 2013), the in vitro transcription assays were completed on naked DNA templates (also see below).Using purified PIC factors, primer extension assays established that transcription initiation occurred at the annotated HSPA1B start site in vitro (Figure S2A), as expected. An overview of the transcription assay is shown in Figure 1A, which was based in part upon in vitro pausing assays with nuclear extracts (Marshall and Price, 1992;Qiu and Gilmour, 2017;Renner et al., 2001). Following PIC assembly, transcription was initiated by adding ATP, GTP, and UTP at physiologically relevant concentrations, with a low concentration of CTP, primarily 32 P-CTP. After one minute, reactions were chased with a physiologically relevant concentration of cold CTP and transcription was allowed to proceed for an additional nine minutes. These "pulse-chase" assays allow better detection of short (potentially paused) transcripts, which otherwise would be drowned out by elongated transcripts that invariably possess more incorporated 32 P-C bases. By directly labeling all transcripts with 32 P-CTP, the method is highly sensitive and allowed detection of transcripts of varied lengths; furthermore, the 32 P-CTP pulse-chase protocol ensured that 32 P-labeled transcripts resulted almost exclusively from single-round transcription (see Methods). Control experiments confirmed that transcripts detected were driven by the HSP70 promoter (e.g. not any contaminating nucleic acid) and that transcription was dependent on added PIC factors, as expected (Figure S2B).A variety of methods have established that RNAPII pauses after transcribing 20-80 bases in Drosophila and mammalian cells (

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“…(D) IPs performed with purified recombinant FACT and purified PAF complex using antibodies to FACT subunits Spt16 and SSRP1 and to the PAF subunit Paf1. initiation competent complex prevents further rounds of transcription by impeding subsequent PIC formation (Hawley and Roeder, 1985;Kraus and Kadonaga, 1998).…”

Section: Effect Of H2bk120ub1 On Single-round Transcription and The Generation Of Longer Transcriptsmentioning

confidence: 99%

Histone H2B Monoubiquitination Functions Cooperatively with FACT to Regulate Elongation by RNA Polymerase II

Pavri

Zhu

et al. 2006

Cell

652

651

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Over the past years, a large number of histone posttranslational modifications have been described, some of which function to attain a repressed chromatin structure, while others facilitate activation by allowing access of regulators to DNA. Histone H2B monoubiquitination is a mark associated with transcriptional activity. Using a highly reconstituted chromatin-transcription system incorporating the inducible RARbeta2 promoter, we find that the establishment of H2B monoubiquitination by RNF20/40 and UbcH6 is dependent on the transcription elongation regulator complex PAF, the histone chaperone FACT, and transcription. H2B monoubiquitination facilitates FACT function, thereby stimulating transcript elongation and the generation of longer transcripts. These in vitro analyses and corroborating in vivo experiments demonstrate that elongation by RNA polymerase II through the nucleosomal barrier is minimally dependent upon (1) FACT and (2) the recruitment of PAF and the H2B monoubiquitination machinery.

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Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II.

Cited by 304 publications

References 26 publications

Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila

Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

Histone H2B Monoubiquitination Functions Cooperatively with FACT to Regulate Elongation by RNA Polymerase II

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