Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex*

Murakami, Kenji; Calero, Guillermo; Brown, Christopher R.; Liu, Xin; Davis, Roger T.; Boeger, Hinrich; Kornberg, Roger D.

doi:10.1074/jbc.m112.433623

Cited by 31 publications

(73 citation statements)

References 29 publications

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“…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).…”

mentioning

confidence: 58%

“…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 addition of Sub1 (yeast homolog of PC4, which stimulates the initiation of transcription) (3,4). The PIC was sedimented in a glycerol gradient in the presence of a nonhydrolyzable analog of ATP.…”

Section: Resultsmentioning

confidence: 99%

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Structure of an RNA polymerase II preinitiation complex

Murakami

Tsai

Kalisman

et al. 2015

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

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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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mentioning

confidence: 58%

Section: Resultsmentioning

confidence: 99%

Structure of an RNA polymerase II preinitiation complex

Murakami

Tsai

Kalisman

et al. 2015

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

Self Cite

109

120

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“…Both isolated RNAPII molecules and stable RNAPII·TFIIF complexes (36,37) were probed; TFIIS was added as indicated. Transcriptional arrest, operationally defined as an event in which a previously transcribing enzyme stalled and no significant net forward translocation occurred over a period of ∼300 s, was consistently observed at hindering loads in the −5 to −20 pN range (SI Appendix, Fig.…”

Section: Tfiif and Tfiis Reactivate Arrested Rnapii By Three Distinctmentioning

confidence: 99%

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms

Schweikhard¹,

Meng

Murakami³

et al. 2014

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

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Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule opticaltrapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.Pol II | optical tweezers | optical trap T he expression of most genes is carefully regulated at the level of transcription. As a consequence, RNA polymerase II (RNAPII)-the enzyme responsible for mRNA synthesis in eukaryotic organisms-is at the nexus of an exquisite network of regulatory pathways, many of which are controlled by transcription factors. The control pathways associated with RNAPII recruitment to, and initiation at, promoter sites have been studied extensively (1), but it has become increasingly clear that significant regulatory activity also occurs during postinitiation steps and, in particular, at the level of transcript elongation (2).Productive transcript elongation (3-5) is characterized by periods of unidirectional motion by RNAPII along the DNA template, adding one nucleotide at a time to the growing RNA transcript. Elongation-both in vitro in highly purified systems (6, 7) and in vivo (8, 9)-is frequently interrupted by transcriptional pauses, at least some fraction of which are associated with enzyme backtracking, a process by which RNAPII reverses its normal direction of motion and moves upstream on the template (6, 7). Entry into backtracked states appears to confer a high degree of for...

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“…The 32-component PIC comprised of TFIIA, TFIIB, TBP, TFIIE, TFIIH, TFIIF, Sub1, and Pol II. The PIC was purified and assembled from proteins that were either available in recombinant form, or were isolated from yeast 21,38 . The hairpin was pulled every 5 min, to allow time for the reassembly of the preinitiation complex between successive pulls.…”

Section: Methodsmentioning

confidence: 99%

Real-Time Observation of Polymerase- Promoter Contact Remodeling during Transcription Initiation

Fazal

Meng

Block

2018

Biophysical Journal

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Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ 70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the −35 and −10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (−10 to −35) and with the −10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.

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Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex*

Cited by 31 publications

References 29 publications

Structure of an RNA polymerase II preinitiation complex

Structure of an RNA polymerase II preinitiation complex

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms

Real-Time Observation of Polymerase- Promoter Contact Remodeling during Transcription Initiation

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