Stepwise mechanism for transcription fidelity

RNA polymerase inhibitors like the CBR class that target the enzyme's complex catalytic center are attractive leads for new antimicrobials. Catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg 2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. We report crystal structures of CBR inhibitor/ Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3′-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.CBR inhibitors | RNA polymerase | transcription inhibition | X-ray crystallography B acteria harbor a single RNA polymerase (RNAP) enzyme (subunit composition α 2 ββ′ω, ∼400 kDa) that performs all transcription in the cell. The bacterial RNAP is a proven target for antimicrobials. The rifamycins (Rifs), which bind to and inhibit the bacterial RNAP (1-3), are potent, broad-spectrum antimicrobials and are the lynchpin of current tuberculosis therapy (4). Nevertheless, the emergence of multidrug-resistant pathogens highlights the importance of discovering novel antimicrobials (5). New RNAP inhibitors can also be used as tools to probe transcriptional mechanisms.Artsimovitch et al. (6) described a new class of bacterial RNAP inhibitors, the CBR compounds. Single amino acid substitutions in the RNAP β and β′ subunits that conferred resistance to CBR compounds (CBR R ) defined a pocket near the RNAP surface bounded by β′ residues of the F loop and the N-terminal part of the bridge helix (BH), and β residues neighboring fork-loop 2 (FL2) and the βDII loop, all RNAP structural elements shown to play important roles in the enzyme's nucleotide addition cycle (NAC) (7-20). The genetically defined CBR site is ∼31 Å from the RNAP active site Mg 2+ ion and is also distinct from the binding sites of other well-characterized bacterial RNAP inhibitors (SI Appendix, Table S1). Inhibition of RNAP by the CBR compounds appears to be mechanistically distinct as well; the CBR compounds allosterically inhibit known catalytic activities of the RNAP active site preferentially at pause sites but have lesser to no effects on translocation (6, 21).The prototype CBR compound [3-(trifluoromethyl)-N-phenylbenzamidoxime, designated CBR703] (Fig. 1 A and B), identified by screening a library of compounds for inhibition of Escherichia coli RNAP, yielded 50% inhibitory co...

Section: Cbr Inhibition Of Rnap Intrinsic Transcript Cleavage Is Tl-dmentioning

confidence: 71%

CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition

Bae

Nayak

Ray

et al. 2015

“…TL residue His1085, for example, interacts with the bound NTP substrate, and is fully conserved among multisubunit RNA polymerases. The importance of this residue is underscored by the lethality of the H1085A substitution in yeast (7) and catalytic defects resulting from substitutions at this position for both the yeast and bacterial enzymes (7,(9)(10)(11). Additionally, a substitution for residue Glu1103 of the TL has been found not only to promote nucleotide misincorporation (6,7), but also to increase the elongation rate (6,7,12), properties that are jointly consistent with the notion of kinetic proofreading (13).…”

mentioning

confidence: 65%

“…If the observed properties of the E1103G substitution were attributable to a stabilization of the closed state of the TL (dependent upon a bound, template-matched NTP), which tends to be more open in the WT, we reasoned that a compensatory destabilization of this closed state might reverse some, or all, of the mutant enzyme properties. Guided by structural data for the TL and RNAPII active site (4, 18), we altered a second residue of the TL, His1085, which, through interaction with NTP substrates, promotes catalysis and likely favors the closed conformation of the TL (4,5,7,(9)(10)(11). H1085A lethality is suppressed by combination with E1103G, but the transcriptional and phenotypic properties of the double substitution suggest mutual suppression (11).…”

Section: Resultsmentioning

confidence: 99%

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Larson

Zhou

Kaplan

et al. 2012

130

187

During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre-or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.optical trap | optical tweezers | Pol II T he transcription of genetic information stably encoded in DNA into a transient RNA message occurs with remarkable fidelity. RNA polymerase (RNAP) incorporates nucleotides into nascent RNA chains at rates of 10-70 s −1 (1, 2), but only inserts the wrong nucleotide approximately once per 100,000 bases, on average (3). Although the energetics of base-pairing is fundamental to transcription fidelity, the discrimination for correctly matched nucleotide substrates is kinetically controlled, and accomplished by active site conformational changes centered on the trigger loop (TL) (4-7). The TL is an evolutionarily conserved mobile element that stabilizes substrate NTPs in the active site of a variety of multisubunit RNA polymerases, including eukaryotic RNAP I, II, and III, as well as bacterial and archaeal RNAP (8). In the case of RNAPII, alteration of the TL has important consequences for activity and fidelity. TL residue His1085, for example, interacts with the bound NTP substrate, and is fully conserved among multisubunit RNA polymerases. The importance of this residue is underscored by the lethality of the H1085A substitution in yeast (7) and catalytic defects resulting from substitutions at this position for both the yeast and bacterial enzymes (7, 9-11). Additionally, a substitution for residue Glu1103 of the TL has been found not only to promote nucleotide misincorporation (6, 7), but also to increase the elongation rate (6,7,12), properties that are jointly consistent with the notion of kinetic proofreading (13). To explore further the interplay between elongation rate and transcriptional fidelity, we determined the effec...

“…Finally, our findings also revealed the key contribution of the 3′-5′ phosphodiester linkage backbone on pol II transcriptional efficiency and fidelity. Previous studies on molecular recognition of RNA pol II have focused on the contributions of functional groups in nucleic acids, such as nucleotide bases, 2′-and 3′-OH of ribose (28)(29)(30)(47)(48)(49)(50). Recently, we used synthetic nucleic acid analogs to reveal the importance of chemical interactions and the intrinsic structural features of nucleic acids in controlling pol II transcriptional fidelity.…”

Section: Key Structural Features Governing Pol II Transcriptional Effmentioning

confidence: 99%

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

Zhang

Chong

et al. 2014

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...