Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

Fishburn, James; Tomko, Eric J.; Galburt, Eric A.; Hahn, Steven

doi:10.1073/pnas.1417709112

Cited by 123 publications

(141 citation statements)

References 37 publications

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“…6A, lanes 5-9). Furthermore, ATP␥S added at 1/3 or 1/9 the concentration of ATP inhibits TFO displacement, with the 1/9 and 1/3 ratios (21). No hydrolysis of either nucleoside triphosphate was observed in the absence of DNA.…”

Section: Resultsmentioning

confidence: 96%

“…Reactions contained a wild-type HIS4 transcription template, purified and recombinant factors as in Fig. 1, and equivalent amounts of holo-TFIIH containing an active (WT) or inactive (E489Q) Ssl2 subunit (21). The inactive variant of Ssl2 was assayed in transcription once.…”

Section: Resultsmentioning

confidence: 99%

“…We next assayed whether GTP, CTP, or UTP can promote displacement of a triple-stranded oligonucleotide by TFIIH, which we showed previously is a measure of TFIIH/Ssl2 DNA translocation (21). In these assays, we utilized individual or combinations of NTPs at 400 M, the levels added to our standard in vitro transcription reactions.…”

Section: Resultsmentioning

confidence: 99%

“…Initial DNA opening requires the Ssl2/XPB (yeast/human) subunit of TFIIH (5,6,20), an ATPdependent DNA translocase (21). Ssl2 is thought to reel downstream DNA into the Pol II cleft, leading to torsional strain and DNA opening (22)(23)(24)(25).…”

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confidence: 99%

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Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Fishburn

Galburt

Hahn

2016

Journal of Biological Chemistry

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Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATP␥S is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATP␥S make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.Following assembly of the metazoan RNA polymerase (Pol) 2 II transcription preinitiation complex (PIC), productive transcription initiation involves at least three steps (1-9): 1) formation of the open complex state containing an ϳ11-base DNA bubble. In this state, the unwound DNA template strand is positioned within the Pol II active site cleft. 2) Synthesis of short RNAs (Յ ϳ9 bases) that initiate within the unwound DNA. This initial transcribing complex can contain a DNA bubble of up to ϳ18 bases resulting from downstream DNA scrunched into the Pol II active site. 3) Promoter escape, where Pol II releases contacts with the basal transcription factors and promoter DNA while transitioning to a processive elongating form. Linked to this release is the collapse of the DNA bubble to the size observed in transcription elongation complexes. Alternatively, during the initial transcription of short RNAs, Pol II can enter a nonproductive state where short abortive RNA products are repeatedly synthesized.Saccharomyces cerevisiae (yeast) Pol II follows a similar but distinct initiation pathway (10). At yeast TATA-containing promoters, most Pol II initiation occurs downstream from the site of PIC formation rather than a fixed distance from the TATA element (11, 12). Initiation typically begins within a window between ϳ50 -100 bases downstream from TATA, with TSS recognition determined in part by the DNA sequence surrounding the TSS (13-16). During the initiation process, yeast Pol II scans downstream DNA for the TSS because Pol II was shown to preferentially utilize the first TSS motif encountered within the 50-to 100-bp window (17). Here we define scanning as the translocation of downstream promoter DNA with respect to Pol II, a process that precedes recognition of the TSS.ATP or dATP hydrolysis is re...

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Fishburn

Galburt

Hahn

2016

Journal of Biological Chemistry

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“…DNA translocation by Ssl2 may therefore lead to promoter melting in the middle (21,22). The DNA-binding sites at the upstream and downstream ends are misaligned, so for simultaneous binding to both sites the DNA must bend.…”

Section: Discussionmentioning

confidence: 99%

Structure of an RNA polymerase II preinitiation complex

Murakami

Tsai

Kalisman

et al. 2015

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

109

120

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The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.-E, -F, and -H, are required for the initiation of RNA polymerase II (pol II) transcription. The GTFs can be assembled with pol II and promoter DNA in a preinitiation complex (PIC) capable of efficient conversion to a transcribing complex (0.1-0.3 transcripts per template). Cryo-EM of the PIC at a resolution of 15-20 Å revealed a bipartite structure, with a P-lobe containing pol II and a G-lobe containing most of the mass of the GTFs (1). A cylinder of electron density, attributed to promoter DNA, was in contact only with GTFs and not with pol II. This architecture was consistent with the pathway of assembly of the PIC, beginning with a complex of promoter DNA and all but one of the GTFs, to which a complex of pol II and the remaining GTF was added (2).A combination of chemical cross-linking and mass spectrometry was used to locate the subunits of the GTFs in the low-resolution cryo-EM electron density map of the PIC. TFIIB was in proximity to promoter DNA at the upstream end of the pol II active center cleft, whereas Ssl2, a subunit of TFIIH, was in apparent contact with promoter DNA at the downstream end of the cleft. TFIIB bridges between pol II and promoter DNA, whereas Ssl2 is a DNA helicase, responsible for unwinding promoter DNA.The association of promoter DNA with the GTFs and not with pol II may be viewed as a fundamental principle of the PIC. Doublestranded DNA is straight and relatively rigid, whereas DNA would need to bend by about 90°to bind in the pol II active center cleft. The GTFs assemble on the double-stranded DNA, position it above the active center, and unwind the DNA. The resulting singlestranded region is flexible and can bend and bind in the pol II cleft.Cryo-EM of the PIC with state-of-the-art instrumentation has resulted in an electron density map at higher resolution. The improved map has confirmed and extended the previous findings and conclusions. ResultsCryo-EM and 3D Reconstruction. A PIC was formed as described (2) on an 86-bp fragment of HIS4 promoter DNA fragment, with pol II, GTFs (TFIIA, TFIIB, TBP, TFIIE, TFIIF, and holo-TFIIH), TFIIS, and with the ad...

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Mitochondrial helicase Irc3 translocates along double‐stranded DNA

et al. 2017

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Irc3 is a superfamily II helicase required for mitochondrial DNA stability in Saccharomyces cerevisiae. Irc3 remodels branched DNA structures, including substrates without extensive single-stranded regions. Therefore, it is unlikely that Irc3 uses the conventional single-stranded DNA translocase mechanism utilized by most helicases. Here, we demonstrate that Irc3 disrupts partially triple-stranded DNA structures in an ATP-dependent manner. Our kinetic experiments indicate that the rate of ATP hydrolysis by Irc3 is dependent on the length of the double-stranded DNA cosubstrate. Furthermore, the previously uncharacterized C-terminal region of Irc3 is essential for these two characteristic features and forms a high affinity complex with branched DNA. Together, our experiments demonstrate that Irc3 has double-stranded DNA translocase activity.

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Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

Cited by 123 publications

References 37 publications

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Structure of an RNA polymerase II preinitiation complex

Mitochondrial helicase Irc3 translocates along double‐stranded DNA

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