Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation

Kireeva, Maria L.; Nedialkov, Yuri A.; Cremona, Gina H.; Purtov, Yuri A.; Lubkowska, Lucyna; Malagón, Francisco; Burton, Zachary F.; Strathern, Jeffrey N.; Kashlev, Mikhail

doi:10.1016/j.molcel.2008.04.017

Cited by 162 publications

(356 citation statements)

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“…The closure of TL is critical for pol II catalytic activity as well as transcriptional fidelity (27)(28)(29)(30)(36)(37)(38)(39). For the wild-type 3′-5′ linkage system, the high transcriptional fidelity is due to pol II's capacity to incorporate matched NTP in a fast and TL-dependent manner (in which TL can achieve a closed conformation during nucleotide addition process) and incorporate mismatched NTP in a slow and TL-independent manner (in which TL remains at an open conformation during nucletotide addition).…”

Section: Discussionmentioning

confidence: 99%

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Zhang

Chong

et al. 2014

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

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Nonenzymatic RNA polymerization in early life is likely to introduce backbone heterogeneity with a mixture of 2′-5′ and 3′-5′ linkages. On the other hand, modern nucleic acids are dominantly composed of 3′-5′ linkages. RNA polymerase II (pol II) is a key modern enzyme responsible for synthesizing 3′-5′-linked RNA with high fidelity. It is not clear how modern enzymes, such as pol II, selectively recognize 3′-5′ linkages over 2′-5′ linkages of nucleic acids. In this work, we systematically investigated how phosphodiester linkages of nucleic acids govern pol II transcriptional efficiency and fidelity. Through dissecting the impacts of 2′-5′ linkage mutants in the pol II catalytic site, we revealed that the presence of 2′-5′ linkage in RNA primer only modestly reduces pol II transcriptional efficiency without affecting pol II transcriptional fidelity. In sharp contrast, the presence of 2′-5′ linkage in DNA template leads to dramatic decreases in both transcriptional efficiency and fidelity. These distinct effects reveal that pol II has an asymmetric (strand-specific) recognition of phosphodiester linkage. Our results provided important insights into pol II transcriptional fidelity, suggesting essential contributions of phosphodiester linkage to pol II transcription. Finally, our results also provided important understanding on the molecular basis of nucleic acid recognition and genetic information transfer during molecular evolution. We suggest that the asymmetric recognition of phosphodiester linkage by modern nucleic acid enzymes likely stems from the distinct evolutionary pressures of template and primer strand in genetic information transfer during molecular evolution.2′-5′ phosphodiester linkage | trigger loop | synthetic nucleic acid analogues T he remarkable capacity of RNA as the functional catalyst as well as the genetic information carrier provides a strong support for the RNA world hypothesis, in which RNA is proposed to play a key role in the early evolution of primitive life before DNA and protein (enzyme) evolved (1, 2). One critical issue for RNA replication in early life is backbone heterogeneity because RNA contains both 2′-OH and 3′-OH, in which both can form phosphodiester bond during RNA polymerization. Indeed, previous studies revealed that nonenzymatic RNA replication would lead to a mixture of 2′-5′ and 3′-5′ linkages (Fig. 1A) (3-9). The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10-15). The 2′-5′ linkage substitutions in some functional RNAs retain molecular recognition and catalytic properties (16)(17)(18)(19). These discoveries suggested that the 2′-5′ linkage in nucleic acids could be an important alternative linkage during early evolution.This backbone heterogeneity problem is largely eliminated during evolution. Modern nucleic acids are dominated by 3′-5′ linkages. The 2′-5′ RNA linkages are only present in a few specific processes. One example is the formation of lariat RNA intron during splicing, where the 2′-OH of an...

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Section: Discussionmentioning

confidence: 99%

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Zhang

Chong

et al. 2014

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

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“…It has been proposed that the TL acts as a critical component of kinetic selection of correctly matched NTP substrates by promoting their faster incorporation in comparison with incorrect substrates (12,30). In support of this hypothesis, substitution of a residue in the ␣-helix at the base of the TL that affected the equilibrium between the closed and open TL conformations decreased fidelity of RNA synthesis by Sce RNA-PII (30). Our results suggest that the F loop also contributes to substrate selection by promoting proper folding of the TL.…”

Section: Discussionmentioning

confidence: 99%

Allosteric control of catalysis by the F loop of RNA polymerase

Miropolskaya

Artsimovitch

Klimašauskas³

et al. 2009

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

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Bacterial RNA polymerases (RNAPs) undergo coordinated conformational changes during catalysis. In particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the RNAP active center have been implicated in nucleotide addition and RNAP translocation. At moderate temperatures, the rate of catalysis by RNAP from thermophilic Thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, Deinococcus radiodurans. Here, we show that a part of this difference is conferred by a third element, the F loop, which is adjacent to the N terminus of the bridge helix and directly contacts the folded trigger loop. Substitutions of amino acid residues in the F loop and in an adjacent segment of the bridge helix in T. aquaticus RNAP for their D. radiodurans counterparts significantly increased the rate of catalysis (up to 40-fold at 20°C). A deletion in the F loop dramatically impaired the rate of nucleotide addition and pyrophosphorolysis, but it had only a moderate effect on intrinsic RNA cleavage. Streptolydigin, an antibiotic that blocks folding of the trigger loop, did not inhibit nucleotide addition by the mutant enzyme. The resistance to streptolydigin likely results from the loss of its functional target, the folding of the trigger loop, which is already impaired by the F-loop deletion. Our results demonstrate that the F loop is essential for proper folding of the trigger loop during nucleotide addition and governs the temperature adaptivity of RNAPs in different bacteria.nucleotide addition ͉ RNA cleavage ͉ streptolydigin ͉ temperature adaptation ͉ transcription C ellular multisubunit RNA polymerases (RNAPs) transcribe genes of up to tens of thousands nucleotides long with high precision and processivity. The catalytic cycle of RNAP is driven by complex conformational changes that accompany NTP binding, catalysis, and RNAP translocation. Recent studies identified two elements in the largest RNAP subunit (␤Ј in bacteria, Rpb1 in eukaryotic RNAP), the trigger loop (TL, or G loop), and the bridge helix (BH, or F-bridge helix), which appear to play the key role during the nucleotide addition cycle (Fig. 1). In the absence of a nucleotide substrate, the TL [amino acids 1234-1254 in Thermus aquaticus (Taq) and Thermus thermophilus RNAPs; amino acids 1077-1097 in Saccharomyces cerevisiae (Sce) RNAPII] adopts an open conformation in which its central part is unstructured (1, 2). Binding of an NTP substrate induces folding of the TL, resulting in extension of the two ␣-helixes at the base of the TL and creating a closed, catalytically competent conformation of the active center in which the NTP is properly aligned with the 3Ј-OH of the nascent RNA for facile catalysis (Fig. 1 A-C) (3, 4). The folded TL and the BH form a three-helix bundle that interacts with the substrate NTP and the template DNA base. In particular, the TL residues M1238 and H1242 (Taq numbering is used throughout the manuscript unless otherwise indicated) contact the base and the phosphate moieties of the ...

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“…1, compare a and b). In addition, Kashlev's and our laboratory showed that the ability of the TL to adopt two conformations is the prerequisite for the high accuracy of RNA synthesis (Kireeva et al, 2008;. In the 'induced fit' mode, the correct NTP induces folding of the TL, which, in turn, provides the physical surface complementary to the NTP and thus stabilizes the transition state of the reaction.…”

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confidence: 99%

Multiple personalities of the RNA polymerase active centre

Zenkin

2014

Microbiology

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Transcription in all living organisms is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Our understanding of the functioning of the active centre of RNAPs has transformed recently with the finding that a conserved flexible domain near the active centre, the trigger loop (TL), participates directly in the catalysis of RNA synthesis and serves as a major determinant for fidelity of transcription. It also appears that the TL is involved in the unique ability of RNAPs to exchange catalytic activities of the active centre. In this phenomenon the TL is replaced by a transcription factor which changes the amino acid content and, as a result, the catalytic properties of the active centre. The existence of a number of transcription factors that act through substitution of the TL suggests that the RNAP has several different active centres to choose from in response to external or internal signals.A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https: //www.youtube.com/watch?v=79Z7iXVEPo4 TRIGGER LOOP: A NEW CATALYTIC DOMAIN OF THE RNA POLYMERASE ACTIVE CENTRE Multi-subunit RNAPs are the enzymes that perform transcription in all living organisms. RNAPs are highly conserved, and emerged before the divergence of the bacteria and archaea/eukaryote lineages. All RNAPs share the invariantly conserved catalytic core of five subunits: b, b9, 2a and v (bacterial nomenclature is used throughout). The two largest subunits, b and b9, form the catalytic cleft where the reaction of addition of nucleoside monophosphates (NMPs) to the growing RNA takes place. As with many other nucleic-acid-managing enzymes (Steitz & Steitz, 1993) and all nucleic-acid-polymerizing enzymes (Steitz, 1998), RNAP uses a two-metal-ion (Mg 2+ ) mechanism to catalyse the phosphotransfer reaction. One of the metal ions, Mg 2+ I, is chelated by the invariant triad of aspartates of the b9 subunit, whilst the other one, Mg 2+ II, is brought by substrates, e.g. by the incoming nucleoside triphosphate (NTP). Mg 2+ I stabilizes the negative charge on the attacking oxygen of the hydroxyl group of the 39 NMP of RNA. Mg 2+ II assists the leaving of the pyrophosphate, and both metal ions ligate the non-bridging oxygen to stabilize the pentacovalent transition state. Substitutions in the aspartate triad lead to almost full inactivation of the enzyme (Zaychikov et al., 1996).The emergence of the first crystal structure of multi-subunit RNAP (Zhang et al., 1999) ignited structure-based functional analysis of the enzyme by many researchers. We were interested in the disordered loop (referred to initially as the G-loop and, later, as the TL) of the highly conserved G domain of the b9 subunit. In the structure, this disordered region was located .20 Å from Mg 2+ I. However, we found that deletion of this loop had a dramatic effect on catalysis by slowing down NMP addition~10 4 times -an effect close to that of mutations in the aspartate triad. Three years later we foun...

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Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation

Cited by 162 publications

References 54 publications

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Allosteric control of catalysis by the F loop of RNA polymerase

Multiple personalities of the RNA polymerase active centre

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