RNA polymerase fidelity and transcriptional proofreading

Sydow, Jasmin F.; Cramer, Patrick

doi:10.1016/j.sbi.2009.10.009

Cited by 144 publications

(144 citation statements)

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“…This observation highlights the importance of the network of contacts that exist in the closed conformation of the enzyme between the incoming NTP and the TL/BH unit and how these interactions can either assist or hinder the folding of the TL necessary for elongation. This mechanism also suggests a role of the TL in fidelity control as has been proposed (4,7,10,34,46,59). Fig.…”

Section: Discussionsupporting

confidence: 68%

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Mejia

Nudler

Bustamante

2014

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

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Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP's pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity. RNA polymerase (RNAP) has been the subject of study for almost five decades by means of a large array of techniques. In the last decade, crystallographic structures of the bacterial and eukaryotic polymerase have allowed researchers to obtain snapshots of the conformational changes that occur deep inside the enzyme, near its catalytic center. Based on these structures, an element, the F-bridge or bridge helix (BH), was first hypothesized to be the essential component in the translocation mechanism of RNAP (1-4). Later, crystal structures of the full elongation complex [RNA, DNA, and nucleoside triphosphate (NTP) bound] identified another structure in close proximity to the BH, termed the trigger loop (TL) (5-7), as another important element in the translocation mechanism. This structure was seen to adopt distinct conformations during the catalytic process, suggesting its role in the kinetic cycle of RNAP. In particular, the TL was seen to contain a dynamic domain that undergoes an unfolding transition during transcription (here termed the TLtip) and another which remains helical throughout the cycle known as the TL base helices. These observations prompted further biochemical characterization of these two structures in their WT form and in variety of point mutants of the BH and TL elements (4,(8)(9)(10)(11)(12).The high-resolution structure of an elongation complex of the Thermus thermophilus polymerase (13) shows the TL in a fully folded helix-turn-helix structure and in close contact with the BH (Fig. S1 A-C). This conformation, in which the TL blocks the secondary channel and correctly positions the incoming NTP for incorporation to occur, has been termed "closed" (10, 14). Based on these structures and other biochemical studies (4,7,8,(15)(16)(17)(18) it has been proposed that NTP binding and the resulting folding of the TL (with its corresponding interactions with the BH) serves as the pawl that rectifies the polymerase's Brownian oscillations on the template by not allowi...

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Section: Discussionsupporting

confidence: 68%

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Mejia

Nudler

Bustamante

2014

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

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“…It was suggested that the elemental pause is characterized by fraying of the 3′-terminal RNA nucleotide away from the DNA template [103]. Structural analysis showed that RNA fraying can occur, as a frayed RNA 3′-nucleotide was observed in a Pol II EC crystal with a mismatched base pair at the end of the hybrid [104,105]. The frayed 3′-nucleotide was found in two alternative sites that overlap with the NTP-binding site, explaining how nucleotide addition is prevented in the elemental pause state.…”

Section: Pausing and Proofreadingmentioning

confidence: 99%

“…2). As a result of structural and biochemical work from several research groups [104,[106][107][108][109][110], a model for transcriptional proofreading has emerged (reviewed in [105]). After nucleotide misincorporation, the mismatched 3′-nucleotide does generally not form a stable nascent base pair at register + 1 but may rather fray away from the DNA template, inducing a paused state and inhibiting RNA extension.…”

Section: Pausing and Proofreadingmentioning

confidence: 99%

Structural basis of transcription elongation

Martínez-Rucobo

Cramer

2013

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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For transcription elongation, all cellular RNA polymerases form a stable elongation complex (EC) with the DNA template and the RNA transcript. Since the millennium, a wealth of structural information and complementary functional studies provided a detailed three-dimensional picture of the EC and many of its functional states. Here we summarize these studies that elucidated EC structure and maintenance, nucleotide selection and addition, translocation, elongation inhibition, pausing and proofreading, backtracking, arrest and reactivation, processivity, DNA lesion-induced stalling, lesion bypass, and transcriptional mutagenesis. In the future, additional structural and functional studies of elongation factors that control the EC and their possible allosteric modes of action should result in a more complete understanding of the dynamic molecular mechanisms underlying transcription elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

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“…Interestingly, GPN proteins have also been demonstrated to interact with the CCT complex, which controls the polymerization of tubulins into microtubules (9). Since the nuclear import of several nuclear proteins has been previously reported to be regulated by the CCT complex, these findings let the authors speculate that there is a possible physiological link between the GPN1 and GPN3 interactions and the import of RNAPII into the nucleus.…”

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confidence: 96%

Regulating the Shuttling of Eukaryotic RNA Polymerase II

Croce

2011

Molecular and Cellular Biology

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The eukaryotic RNA polymerase II (RNAPII) transcribes most protein-coding RNAs (mRNAs) and the capped noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) (10). In the past 3 decades, our knowledge about the functional role of RNAPII during gene transcription has dramatically advanced (8). However, there are still several open questions regarding the regulatory mechanisms involved in the steps before and after transcription. In particular, how RNAPII is imported into the nucleus remains elusive. In this issue, Carré and Shiekhattar (3) report that the evolutionarily conserved GTPases GPN1 and GPN3 stably associate with and regulate the nuclear import of the human RNAPII. These exciting results now make it possible to begin to dissect the regulatory pathways of the intracellular localization and nuclear import of RNAPII.Recently, the structures and subunit compositions of the three RNA polymerases (Table 1) have been characterized (4). RNAPII is a highly conserved enzyme composed of 12 subunits, 10 of which form the central core. The other two subunits (Rpb4 and Rpb7) form a peripheral heterodimer that protrudes out from the core enzyme structure. This stabilizes the RNAPII complex in clamp-closed conformation, favoring efficient transcription initiation. Rpb10 and Rpb12 are common subunits for all of the eukaryotic RNA polymerases and are implicated in the interaction with the basal transcription factors, such as the TATA box-binding protein (TBP). These interactions lead to the formation of the preinitiation complex (PIC), which contains RNAPII as well as the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. In the past years, numerous coactivators that both facilitate the access of PIC to its target promoters and increase the stability of the complex have been functionally characterized. Recent data (1) indicate that the RNAPII assembly pathway occurs in the cytoplasm and that assembly is coordinated by the consecutive actions of the cochaperone hSpagh and the chaperone Hsp90. The two largest subunits, Rpb1 and Rpb2, are initially preassembled into two separated subcomplexes, together with several other subunits that are either common to all three RNA polymerases or are specific for RNAPII (Fig. 1). It is likely that the two subcomplexes are then assembled together before they enter the nucleus through the nuclear pore complex (NPC). Interestingly, it now seems that access to the nucleus is restricted exclusively to the assembled RNAPII, thus preventing any premature binding of the independent subunits at promoter regions.But the question of how the assembled RNAPII is imported into the nucleus remains. How is this step regulated? Carré and Shiekhattar tackled this question using a combination of cell biology approaches and mass spectrometry of affinity-purified complexes containing tagged cytoplasmic and nuclear RNAPII (3). They identified GPN1 and GPN3 as novel interactors of the RNAPII, which remained associated with...

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RNA polymerase fidelity and transcriptional proofreading

Cited by 144 publications

References 90 publications

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Structural basis of transcription elongation

Regulating the Shuttling of Eukaryotic RNA Polymerase II

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