Analysis of RNA Polymerase II Elongation In Vitro

Adamson, Todd E.; Shore, Sarah M.; Price, David H.

doi:10.1016/s0076-6879(03)71019-2

Cited by 28 publications

(57 citation statements)

References 27 publications

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“…TFIIF is an established initiation and elongation factor (54 -56), and we exploited its elongation properties by adding it to the chase step where it efficiently promoted elongation (compare lanes 1 and 2, Fig. 1C) (46). To this TFIIF-dependent elongation assay we titrated rOGA, and we found a clear reduction in elongation (lanes 3-5, Fig.…”

Section: Oga Activity Is Required For Efficient Elongation In Vitro-mentioning

confidence: 94%

“…To address a possible OGA function during elongation, we adopted the elongation assay established by Price and co-workers (46). We used nuclear extracts made from HeLa cells (45) and assayed transcription activity using the CMV promoter and a pulse-chase assay (46).…”

Section: Oga Activity Is Required For Efficient Elongation In Vitro-mentioning

confidence: 99%

“…We used nuclear extracts made from HeLa cells (45) and assayed transcription activity using the CMV promoter and a pulse-chase assay (46). We used this assay to test various kinase and OGA inhibitors specifically for effects on elongation by adding the inhibitor(s) after allowing PIC formation to occur for 30 min.…”

Section: Oga Activity Is Required For Efficient Elongation In Vitro-mentioning

confidence: 99%

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O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β

Resto

Kim

Fernandez

et al. 2016

Journal of Biological Chemistry

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We describe here the identification and functional characterization of the enzyme O-GlcNAcase (OGA) as an RNA polymerase II elongation factor. Using in vitro transcription elongation assays, we show that OGA activity is required for elongation in a crude nuclear extract system, whereas in a purified system devoid of OGA the addition of rOGA inhibited elongation. Furthermore, OGA is physically associated with the known RNA polymerase II (pol II) pausing/elongation factors SPT5 and TRIM28-KAP1-TIF1␤, and a purified OGA-SPT5-TIF1␤ complex has elongation properties. Lastly, ChIP-seq experiments show that OGA maps to the transcriptional start site/5 ends of genes, showing considerable overlap with RNA pol II, SPT5, TRIM28-KAP1-TIF1␤, and O-GlcNAc itself. These data all point to OGA as a component of the RNA pol II elongation machinery regulating elongation genome-wide. Our results add a novel and unexpected dimension to the regulation of elongation by the insertion of O-GlcNAc cycling into the pol II elongation regulatory dynamics.RNA polymerase II is the species of polymerase that transcribes the protein-coding genes in the cell. Largely based on in vitro transcription data and bacterial transcriptional regulation, it was thought that the pathway of transcription initiation was the de novo assembly and recruitment of general factors and pol II 4 into a preinitiation complex at promoters. In other words, transcription was solely controlled by whether initiation occurred or not. However, in the late 1980s, a transcriptionally engaged pol II was found on several genes, located roughly at ϩ50 relative to the transcriptional start site (TSS) (1-3). More recently, genome-wide approaches have shown that paused pol II exists on at least 40% of promoters in the genome (4 -9).These data show that the regulation of elongation is a significant and widespread method by which cells regulate gene expression.Biochemically, the establishment of a paused polymerase requires the recruitment of DSIF and NELF to pol II early in elongation to prevent pol II from productive elongation (10 -13). Release of the paused polymerase is achieved with P-TEFb phosphorylation of DSIF and NELF, ejecting NELF from pol II and converting DSIF into a positive elongation factor (14,15). The more recent discoveries of additional factors likely involved in pause establishment and release include human capping enzyme (16), the TFIIH-associated kinase, CDK7 (17, 18), the TFIIH ERCC3 helicase (19), , Integrator (23,24), ELL (25), TFIIS (26), TRIM28-KAP1-TIF1␤ (27, 28), Top1 (29), SEC (30), PAF complex (31, 32), and Gdown1 (33). The plethora of factors suggests that more complicated dynamics are at play in regulating pausing and elongation. Additionally, it is not clear, and indeed difficult to know, whether all of the factors involved in pol II pausing and elongation have been identified. This difficulty is due mostly to the absence of a human cell-based in vitro transcription system that recapitulates a paused polymerase and with which the full complement of...

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Section: Oga Activity Is Required For Efficient Elongation In Vitro-mentioning

confidence: 94%

Section: Oga Activity Is Required For Efficient Elongation In Vitro-mentioning

confidence: 99%

Section: Oga Activity Is Required For Efficient Elongation In Vitro-mentioning

confidence: 99%

See 1 more Smart Citation

O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β

Resto

Kim

Fernandez

et al. 2016

Journal of Biological Chemistry

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“…After the pulse, the EECs were repeatedly washed with 1 M KCl and 1% Sarkosyl to strip all initiation and elongation factors from the elongation complex (28). HNE was added back to the isolated EECs and elongation continued (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In Vitro Transcription Assays-HeLa nuclear extract (HNE) preparation and immobilized template pulse-chase transcriptions were performed essentially as described previously (28). The usual transcription reaction was 12 l and contained 20 mM HEPES, 60 mM KCl, 5 mM MgCl 2 , 1 mM dithiothreitol, 100 M S-adenosylmethionine, 1 unit/l of RNaseOUT (Invitrogen), 1.5 l of HNE, and 20 ng/l of template.…”

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confidence: 99%

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Adamson

Shutt

Price

2005

Journal of Biological Chemistry

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Cleavage and polyadenylation define the 3 ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b (P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5 portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5 capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.Processing of eukaryotic mRNA starts during transcription and is influenced by the RNA polymerase II elongation complex (1, 2). Capping, polyadenylation, and splicing have been seen to occur on nascent transcripts in vitro, and a variety of in vivo and in vitro approaches have strongly implicated the carboxyl-terminal domain (CTD) 2 of the large subunit of RNA polymerase II in connecting transcription with these events (3-6). Whereas many of the factors required for mRNA processing have been identified and characterized, much less is known about how the processing and transcription machinery functionally influence each other.3Ј End formation is linked to transcription. Although RNA polymerase II is capable of transcribing hundreds of kilobase pairs in a completely processive manner, after transcribing a functional polyadenylation signal the polymerase usually terminates within 1 kb (3, 7) in a process that requires the CTD (8, 9). Factors required for polyadenylation have been found to associate with isolated transcription complexes (10) and the CTD has been implicated in bringing in the factors (11, 12). There is some controversy about when polyadenylation factors associate with the transcription complex. Polyadenylation factors have been found to associate with promoter binding factors (13), and yeast chromatin immunoprecipitation experiments in one study have localized the factors throughout the gene (11), but in another only at the 3Ј end of genes after the passage of a functional polyadenylation signal (14). Recent studies in yeast (15), Drosophila (16), and Xenopus (17) have implicated the CTD kinase positive transcription elongation factor b (P-TEFb) in promoting efficient polyadenylation. Drosophila histone mRNA 3Ј end formation, involving a completely different set of factors, was found not to be stimulated by having the RNA in an elongation complex (18). However, a strong transcriptional pause was found a...

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Quantitative analysis of EDC‐condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

Mann

McKnight

Melechko

et al. 2007

Biotech & Bioengineering

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Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. Polymerase chain reaction (PCR) and in vitro transcription (IVT) were used to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. This investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

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Analysis of RNA Polymerase II Elongation In Vitro

Cited by 28 publications

References 27 publications

O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β

O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Quantitative analysis of EDC‐condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

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