The Role of the Transcription Bubble and TFIIB in Promoter Clearance by RNA Polymerase II

Pal, Mahadeb; Ponticelli, Alfred S.; Luse, Donal S.

doi:10.1016/j.molcel.2005.05.024

Cited by 123 publications

(187 citation statements)

References 44 publications

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“…Consistent with this, the B-reader of the archaeal TFIIB homologue stimulates transcription 22 ; truncation of the B-reader loop in human TFIIB impairs extension of a RNA dinucleotide 23 ; and the presence of TFIIB and TBP stimulates transcription initiation from pre-opened DNA 15,24 . We find here that TFIIB alone stimulates de novo RNA synthesis by Pol II at low or near-physiological NTP concentrations ( Fig.…”

mentioning

confidence: 74%

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Sainsbury

Niesser

Cramer

2012

Nature

179

195

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The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II-TFIIB complex 1,2 indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions 3,4 enigmatic. Here we report crystal structures of the Pol II-TFIIB complex from the yeast Saccharomyces cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The structures reveal the entire B-reader and proteinnucleic acid interactions, and together with functional data lead to a more complete understanding of transcription initiation. TFIIB partially closes the polymerase cleft to position DNA and assist in its opening. The B-reader does not reach the active site but binds the DNA template strand upstream to assist in the recognition of the initiator sequence and in positioning the transcription start site. TFIIB rearranges active-site residues, induces binding of the catalytic metal ion B, and stimulates initial RNA synthesis allosterically. TFIIB then prevents the emerging DNA-RNA hybrid duplex from tilting, which would impair RNA synthesis. When the RNA grows beyond 6 nucleotides, it is separated from DNA and is directed to its exit tunnel by the B-reader loop. Once the RNA grows to 12-13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors 5 , and the bacterial initiation factor sigma has TFIIB-like topology 1,2 and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function [6][7][8] . TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.Our previous X-ray analysis of the Pol II-TFIIB complex at 4.3 Å resolution provided a partial backbone model of TFIIB 1 . To obtain a complete and atomic structure, we co-crystallized Pol II with a TFIIB variant lacking the mobile amino-terminal tail and carboxy-terminal cyclin fold (Methods, Supplementary Table and Supplementary Fig. 1). The resulting Pol II-TFIIB structure at 3.4 Å resolution provides details of the interactions of the four TFIIB domains with Pol II: the B-ribbon with the dock, the B-core N-terminal cyclin fold with the wall, the B-reader helix with the RNA exit tunnel, and the B-linker helix with the coiled-coil of the clamp (Fig. 1).The structure reveals the entire course of the TFIIB polypeptide chain through the Pol II cleft, including the previously lacking 1 B-reader loop (residues 67-79) and a new 'B-reader strand' (residues 80-83). The B-reader loop does not reach the active site, but instead interacts with the Pol II rudder and fork loop ...

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mentioning

confidence: 74%

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Sainsbury

Niesser

Cramer

2012

Nature

179

195

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“…Although useful in engaging a wide variety of foreign DNA, this intrinsically weak intermolecular force does not generate enough binding energy beyond formation of an encounter complex (34). Thus, despite exceeding the footprint of the HIN200 domains, the length of the exposed linker-dsDNA between nucleosomes [10-20 bp (21)] or even that of the transcription bubble [∼17 bases (22)] is too short to promote robust filament assembly of IFI16. Additionally, we envision that requiring oligomerization to achieve tight binding also plays a negative role in competing against replication/transcription machinery.…”

Section: Discussionmentioning

confidence: 99%

“…For instance, AIM2 has only one HIN200 domain and is exclusively localized in the cytoplasm (19). By contrast, IFI16 has two HIN200 domains that can bind either ss-or dsDNA and assumes an uninhibited open conformation, and its footprint falls within the range of the exposed dsDNA linker between host nucleosomes (10-20 bp) or transcription bubbles (∼17 bases) (12,(20)(21)(22). Indeed, the proposed autoinhibitory mechanism would be effective only in an intrinsically DNA-free environment like the cytoplasm but not in the nucleus, where abundant self-DNA would easily displace IFI16 PYD from either HIN200 domain.…”

Section: Significancementioning

confidence: 99%

Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy

Morrone

Wang²,

Constantoulakis³

et al. 2013

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

153

209

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Whether host DNA receptors have any capacity to distinguish self from nonself at the molecular level is an outstanding question in the innate immunity of mammals. Here, by using quantitative assays and electron microscopy, we show that cooperatively assembling into filaments on dsDNA may serve as an integral mechanism by which human IFN-inducible protein-16 (IFI16) engages foreign DNA. IFI16 is essential for defense against a number of different pathogens, and its aberrant activity is also implicated in several autoimmune disorders, such as Sjögren syndrome. IFI16 cooperatively binds dsDNA in a length-dependent manner and clusters into distinct protein filaments even in the presence of excess dsDNA. Consequently, the assembled IFI16·dsDNA oligomers are clearly different from the conventional noninteracting entities resembling beads on a string. The isolated DNA-binding domains of IFI16 engage dsDNA without forming filaments and with weak affinity, and it is the non-DNA-binding pyrin domain of IFI16 that drives the cooperative filament assembly. The surface residues on the pyrin domain that mediate the cooperative DNA binding are conserved, suggesting that related receptors use a common mechanism. These results suggest that IFI16 clusters into signaling foci in a switch-like manner and that it is capable of using the size of naked dsDNA as a molecular ruler to distinguish self from nonself.cooperative filament formation | inflammasome R ecognition of foreign intracellular DNA is a widely conserved defense mechanism by which the innate immune system of mammals detects and responds to invading pathogens (1, 2). Using such a universal molecule as a major danger signal for detecting pathogens must be regulated in a stringent yet efficient manner. However, only a few deciding factors are known for the host innate immune system to selectively engage foreign DNA (i.e., nonself-DNA) while minimizing interactions with self-DNA, which include the compartmentalization of mammalian cells and the size of foreign DNA. These features, however, only raise more questions than provide answers. For example, the footprints of intracellular DNA receptors usually fall below 20 bp, and yet a long foreign DNA fragment [e.g., poly(dA:dT); ≥1,000 bp] is required to induce a robust innate immune response even in a normally DNA-free environment like the cytoplasm (1, 2). On the other hand, foreign DNA-sensing pathways also exist in the host nucleus in which DNA receptors must not respond to abundant self-DNA to prevent spurious activities (1, 2). Indeed, one of the major unresolved questions in understanding the DNA-sensing pathways of mammals is whether the host intracellular DNA receptors have any capacity to distinguish self-from nonself-DNA at the molecular level (2).Human IFN inducible protein-16 (IFI16) is an intracellular DNA receptor of innate immunity that belongs to the family of absent-in-melanoma-2 (AIM2)-like receptors (ALRs) (1-4). IFI16 senses DNA from invading pathogens in both the nucleus and cytoplasm [e.g., vaccinia virus...

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“…This primary effect of DSIF may be amplified in steady-state transcription conditions where committed templates would be prevented from freely reinitiating perhaps owing to the promoter-proximally stalled Pol II. Potentially related, and in view of the possibility that TFIIB likely has multiple roles in the transcription cycle (3,33), the ability of TFIIB to bypass the Mediator requirement may also reflect additional functions at the postinitiation level.…”

Section: Discussionmentioning

confidence: 99%

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Malik

Barrero

Jones

2007

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

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The multiprotein Mediator coactivator complex is universally required for transcription of metazoan genes. It has been proposed to function by interfacing between transcriptional activators and the RNA polymerase II machinery. However, in vitro transcription systems reconstituted from homogeneous preparations of RNA polymerase II, the general transcription initiation factors, and the cofactor PC4 display relatively robust activator (HNF-4)-dependent activity, which, nonetheless, can be further stimulated by Mediator. By contrast, an unfractionated nuclear extract-based system in which Mediator has been immunodepleted displays a nearabsolute dependence on ectopic Mediator. Here, we identified and purified an activity, MSA-2, that confers extract-like Mediator responsiveness to our reconstituted system. Mass spectrometric analyses identified its two constituent polypeptides as hSpt5 and hSpt4, which also comprise the elongation factor DSIF. Mechanistically, MSA-2/DSIF acts by restricting overall transcription in the pure system, thereby imposing a strong Mediator dependence. Our data thus point to potential mechanisms for Mediator function beyond its presently believed role in promoting the initial formation of the RNA polymerase II-containing preinitiation complex.DRB sensitivity-inducing factor ͉ hepatocyte nuclear factor-4 ͉ positive cofactor 2 ͉ RNA polymerase II

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The Role of the Transcription Bubble and TFIIB in Promoter Clearance by RNA Polymerase II

Cited by 123 publications

References 44 publications

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

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