Structure of an RNA polymerase II–RNA inhibitor complex elucidates transcription regulation by noncoding RNAs

Kettenberger, Hubert; Eisenführ, Alexander; Brueckner, Florian; Theis, Mirko; Famulok, Michael; Cramer, Patrick

doi:10.1038/nsmb1032

Cited by 70 publications

(71 citation statements)

References 23 publications

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“…In particular, high-salt crystallization conditions (15) and/or crystal packing interactions may disfavor binding of 1.1 in the channel, and this or other aspects of crystallization may induce the ''narrow'' conformation of flexible/ mobile elements of the enzyme. The results reported here and the publication of a crystal structure of a dsRNA ligand bound in the structurally homologous cleft of the yeast RNAP (24) demonstrate that double-stranded nucleic acids can indeed access the channel, ruling out a general requirement for DNA opening to precede entry.…”

Section: Comparison Of the Extent Of Dna Opening In Rpo With I1 Usingmentioning

confidence: 62%

“…At 17°C, where the equilibrium constant K 1 is a maximum and conversion of I 1 to I 2 is relatively slow, we previously found that the fraction of promoter DNA in I 1 complexes ( I1 ) at 15 sec after mixing P R promoter with 70 nM (excess) RNAP was 0.55 (6). To increase the fraction of I 1 in the time interval (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) (38) where the only complex predicted to be populated is I 1 (9). Because the signal:noise ratio in these 0°C experiments was marginal, and to avoid the possibility of populating off-pathway complexes at 0°C (39), we identified solution conditions where I 1 is Ͼ70% of promoter DNA (see above) at early times in the time course of RP o formation, and footprinted as follows.…”

Section: Determination Of Fractions Of Promoter Dna In I1 and Rpo Commentioning

confidence: 99%

See 1 more Smart Citation

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Davis¹,

Bingman

Landick³

et al. 2007

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

197

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initiation ͉ transcription ͉ wrapping ͉ kinetics S pecific transcription initiation by Escherichia coli RNA polymerase (RNAP: core subunit composition ␣ 2 ␤␤Ј ϩ 70 ϭ holoenzyme) at promoter sequences is determined by recognition of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) by the specificity subunit 70 . Subsequent to binding, a series of large-scale conformational changes in both RNAP and promoter DNA create the initiation-competent open complex (RP o ) (1). During these steps, the multisubunit bacterial RNAP acts as an intricate molecular machine and opens Ϸ14 bp of the DNA double helix. Defining the cascade of conformational changes that occur during initiation is essential to understand sequence-and factordependent regulation of the rate of transcription initiation and has important applications in chemical biology and in antibiotic design. However, the intermediates on this pathway are relatively unstable and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RP o formation has come from chemical and enzymatic DNA footprinting methods.Quantitative kinetic-mechanistic studies find that at least two kinetically significant intermediates, generically designated I 1 and I 2 , precede formation of RP o by E. coli RNAP:where the relatively slow interconversions between I 1 and I 2 are rate-limiting in both the forward and back directions (2, 3). In the mechanism shown in Eq. 1, I 2 and RP o are characterized by their resistance to a short challenge with a polyanionic competitor such as heparin, which acts to sequester any free RNAP present during the challenge. (In contrast, after a 10 to 20 sec challenge with heparin, I 1 complexes, which are in rapid equilibrium with free RNAP and promoter sequences, are eliminated from the population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely to describe the key steps in initiation in most prokaryotes. Moreover, conservation of many elements of sequence, structure, and/or function between bacterial and eukaryotic polymerase (pol II) subunits and transcription factors supports the inference that the bacterial intermediates may be homologs of initiation intermediates formed by pol II (4, 5).Recently we (6) and Ross and Gourse (7) found that the presence of DNA upstream of the Ϫ35 promoter recognition hexamer greatly accelerates (up to Ϸ60-fold) the rate-determining isomerization step (conversion of I 1 to I 2 ). Strikingly, DNase I footprinting of I 1 at the strong bacteriophage promoter P R reveals that when nonspecific DNA upstream of base pair Ϫ47 is present, downstream DNA is protected to around ϩ20, and thus bound in the active-site channel of RNAP. However, when DNA upstream of Ϫ47 is deleted,

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Section: Comparison Of the Extent Of Dna Opening In Rpo With I1 Usingmentioning

confidence: 62%

Section: Determination Of Fractions Of Promoter Dna In I1 and Rpo Commentioning

confidence: 99%

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Davis¹,

Bingman

Landick³

et al. 2007

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

197

View full text Add to dashboard Cite

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“…Therefore, our data support a model in which it is the repression domains of B2 RNA and Alu RNA that exclude polymerase-promoter contacts. A crystal structure of a synthetic RNA aptamer that binds yeast Pol II and represses transcription shows that a region of the aptamer forms a double stem-loop structure in the DNA binding cleft of Pol II, which would preclude the template DNA strand from entering the cleft (20). The data presented here are consistent with a model whereby the repression domains of B2 and Alu RNAs block transcription by interacting with the DNA cleft of Pol II in a manner similar to the synthetic aptamer.…”

Section: Discussionmentioning

confidence: 99%

B2 RNA and Alu RNA repress transcription by disrupting contacts between RNA polymerase II and promoter DNA within assembled complexes

Yakovchuk

Goodrich

Kugel

2009

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

146

135

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Noncoding RNAs (ncRNAs) are now recognized as transregulators of eukaryotic transcription, a role once attributed exclusively to protein factors. Two ncRNAs in mammalian cells have been shown to repress general mRNA transcription by RNA polymerase II (Pol II) in response to heat shock: mouse B2 RNA and human Alu RNA. B2 and Alu RNAs bind directly and tightly to Pol II and co-occupy the promoters of repressed genes along with the polymerase. Here, we identified the molecular mechanism by which mouse B2 RNA and human Alu RNA repress Pol II transcription. Biochemical assays to probe the network of protein-DNA interactions at the promoter revealed that B2 and Alu RNAs prevent Pol II from establishing contacts with the promoter both upstream and downstream of the TATA box during closed complex formation. Disruption of these contacts correlates with transcriptional repression. We conclude that B2 and Alu RNA prevent Pol II from properly engaging the DNA during closed complex formation, resulting in complexes with an altered conformation that are transcriptionally inert. In the absence of its normal contacts with the promoter, Pol II is likely held in these inactive complexes on DNA through interactions with promoter-bound TATA box-binding protein and transcription factor IIB.T ranscription is an intricate biological process in which DNA is copied into RNA; it is the critical first step in gene expression. In eukaryotes, the enzyme RNA polymerase II (Pol II) transcribes protein-encoding genes into mRNA with assistance of general transcription factors (GTFs; specifically TFIIA, TFIID, TFIIB, TFIIF, TFIIE, and TFIIH) that are thought to function at most promoters (1). Transcriptional regulation of specific genes occurs through the remarkably balanced interplay of auxiliary factors such as promoter-specific activators and repressors, coregulators, and chromatin-modifying complexes (1). Traditionally, it was thought that all of these factors were proteins, but now it is becoming clear that noncoding RNAs (ncRNAs) also play important roles in regulating transcription. Indeed, diverse ncRNAs have been identified as regulators of nearly every step in the process of mRNA transcription from controlling chromatin structure through regulating transcript elongation (2).Our laboratory reported that 2 ncRNAs, mouse B2 RNA and human Alu RNA, repress mRNA transcription by binding to Pol II during the cellular heat shock response (3, 4). B2 and Alu RNAs are transcribed by RNA polymerase III from short interspersed elements (SINEs) (5). Upon heat shock, the levels of B2 RNA and Alu RNA increase (6, 7) and they function as general repressors of mRNA transcription (3, 4). Biochemical experiments showed that B2 and Alu RNAs bind directly to core Pol II with low nM affinity (4, 8). Other SINE RNAs have been identified in mammalian cells, including mouse B1 RNA and human scAlu RNA (5), the latter of which is likely derived from cleavage of full-length Alu RNA (9). The biological functions for B1 and scAlu RNAs are not known. In vitro both of...

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“…In addition to dimerization, hibernation implies cleft expansion and ordering of the DNA-mimicking loop inside the cleft. In Pol II and bacterial RNA polymerase, it was shown that certain RNAs and proteins can block the enzyme by binding inside the cleft [36–38]. The DNA-mimicking loop within the expander could have a protective function in the Pol I hibernating state, by hampering the binding of macromolecules that could compromise enzyme reactivation.…”

Section: Pol I Hibernation By Dimerizationmentioning

confidence: 99%

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

Fernández‐Tornero

2018

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Structure of an RNA polymerase II–RNA inhibitor complex elucidates transcription regulation by noncoding RNAs

Cited by 70 publications

References 23 publications

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

B2 RNA and Alu RNA repress transcription by disrupting contacts between RNA polymerase II and promoter DNA within assembled complexes

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

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