Transcriptional Pausing at +62 of the HIV-1 Nascent RNA Modulates Formation of the TAR RNA Structure

Palangat, Murali; Meier, Timothy I.; Keene, Richard G.; Landick, Robert

doi:10.1016/s1097-2765(00)80103-3

Cited by 101 publications

(116 citation statements)

References 58 publications

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“…DNA templates with upstream biotin tags were prepared by PCR amplification of plasmid pLL283 or pPM151A (truncated HIV-1 LTR) and immobilized on streptavidinagarose beads (Sigma), as described (27,28). pPM151A was derived from pLL283 by deleting DNA coding for A15 through G48 of the HIV-1 transcript (template 2; Fig.…”

Section: Methodsmentioning

confidence: 99%

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A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

Palangat

Renner

Price

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-␤-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF͞ NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF͞NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF͞NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires >18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of backtracked nascent RNA, TFIIS inhibition may help DSIF͞NELF negatively regulate productive transcription.transcription elongation ͉ pausing ͉ backtracking R egulation of productive mRNA chain synthesis by RNA polymerase II (RNAPII) occurs when the RNA chain is Ϸ10-100 nt long and determines whether RNAPII forms a fully functional transcription elongation complex (TEC) or halts RNA synthesis during the early stages of transcript elongation (reviewed in refs. 1-5). Conversion to a productive TEC involves an ordered set of transitions that require successive phosphorylation of Ser-5 and Ser-2 in the RPB1 C-terminal heptapeptide repeat domain (CTD) by kinase components of TFIIH and positive elongation factor P-TEFb, respectively. These CTD changes orchestrate release of initiation factors, recruitment of general elongation and chromatin-modifying factors, and capping of the nascent transcript by the capping enzyme. Completion of these steps is required to produce a TEC able to transcribe through nucleosomes on a chromatin template and synthesize full-length pre-mRNAs.

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Section: Methodsmentioning

confidence: 99%

“…U14 TECs were prepared, converted to TECs with longer RNAs, and used for transcription assays as described. (27). Pause strengths (Fig.…”

Section: Methodsmentioning

confidence: 99%

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

Palangat

Renner

Price

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…After misincorporation, the mismatched nucleotide at position +1 of the RNA is frayed away from the template, thereby pausing RNAP. Pausing is the first step in backtracking [63,[76][77][78][79][80][81][82][83]. The frayed nucleotide then inhibits RNA extension, because it prevents NTP binding, but favors backtracking, because the bp in position +1 is disrupted.…”

Section: Model For Transcriptional Proofreadingmentioning

confidence: 99%

RNA polymerase fidelity and transcriptional proofreading

Sydow

Cramer

2009

Current Opinion in Structural Biology

144

133

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Whereas mechanisms underlying the fidelity of DNA polymerases (DNAPs) have been investigated in detail, RNA polymerase (RNAP) fidelity mechanisms remained poorly understood. New functional and structural studies now suggest how RNAPs select the correct nucleoside triphosphate (NTP) substrate to prevent transcription errors, and how the enzymes detect and remove a misincorporated nucleotide during proofreading. Proofreading begins with fraying of the misincorporated nucleotide away from the DNA template, which pauses transcription. Subsequent backtracking of RNAP by one position enables nucleolytic cleavage of an RNA dinucleotide that contains the misincorporated nucleotide. Since cleavage occurs at the same active site that is used for polymerization, the RNAP proofreading mechanism differs from that used by DNAPs, which contain a distinct nuclease specific active site.

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“…Transcriptional pausing can not only reduce rates of mRNA production, but also recruit regulatory factors to the TEC that modify subsequent transcription (109)(110)(111)(112), function as a precursor to transcriptional arrest and termination (113,114), help to synchronize transcription and translation in prokaryotes (115), or lead to messenger splicing or polyadenylation in eukaryotes (116,117). The longlived, "stabilized pauses" that are known to play a regulatory role are often associated with the formation of RNA hairpins in the transcript (which are thought to allosterically inactivate RNAP), or with the formation of energetically weak RNA:DNA hybrids (which are thought to induce backtracking) (118).…”

Section: Off-pathway Eventsmentioning

confidence: 99%

Single-Molecule Studies of RNA Polymerase: Motoring Along

Herbert

Greenleaf

Block

2008

Annu. Rev. Biochem.

187

141

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Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

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Transcriptional Pausing at +62 of the HIV-1 Nascent RNA Modulates Formation of the TAR RNA Structure

Cited by 101 publications

References 58 publications

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

RNA polymerase fidelity and transcriptional proofreading

Single-Molecule Studies of RNA Polymerase: Motoring Along

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