The nucleotide addition cycle of the SARS-CoV-2 polymerase

Background: The ability of Pseudouridimycin (PUM) to occupy the nucleotide addition site of bacterial RNA Polymerase (RNAP) underlies its inhibitory potency as previously reported. PUM has gained high research interest as a broad-spectrum nucleoside analog that has demonstrated exciting potentials in treating drug-resistant bacterial infections. Objective: Herein, we identified, for the first time, a novel complementary mechanism by which PUM elicits its inhibitory effects on bacterial RNAP. Methods: The dynamic binding behavior of PUM to bacterial RNAP was studied using various dynamic analyses approaches. Results and Discussion: Findings revealed that in addition to occupying the nucleotide addition site, PUM also interrupts the unimpeded entry and exit of DNA by reducing the mechanistic extension of the RNAP cleft and perturbing the primary conformations of the switch regions. Moreover, PUM binding reduced the distances between key residues in the β and β’ subunits that extend to accommodate the DNA. Conclusion: This study’s findings present structural insights that would contribute to the structure-based design of potent and selective PUM inhibitors.

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THEORETICAL INVESTIGATIONS ON ELUCIDATING FUNDAMENTAL MECHANISMS OF CATALYSIS AND DYNAMICS INVOLVED IN TRANSCRIPTION BY RNA POLYMERASE

Journal of Theoretical and Computational Chemistry ◽

10.1142/s0219633613410058 ◽

2013 ◽

Vol 12 (08) ◽

pp. 1341005 ◽

Cited By ~ 4

Author(s):

FÁTIMA PARDO-AVILA ◽

LIN-TAI DA ◽

YING WANG ◽

XUHUI HUANG

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Normal Mode Analysis ◽

Md Simulations ◽

Mode Analysis ◽

Transcription Process ◽

Nucleotide Addition ◽

Theoretical Investigations ◽

State Models ◽

Elongation Process

RNA polymerase is the enzyme that synthesizes RNA during the transcription process. To understand its mechanism, structural studies have provided us pictures of the series of steps necessary to add a new nucleotide to the nascent RNA chain, the steps altogether known as the nucleotide addition cycle (NAC). However, these static snapshots do not provide dynamic information of these processes involved in NAC, such as the conformational changes of the protein and the atomistic details of the catalysis. Computational studies have made efforts to fill these knowledge gaps. In this review, we provide examples of different computational approaches that have improved our understanding of the transcription elongation process for RNA polymerase, such as normal mode analysis, molecular dynamic (MD) simulations, Markov state models (MSMs). We also point out some unsolved questions that could be addressed using computational tools in the future.

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Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides

Nucleic Acids Research ◽

10.1093/nar/gkf547 ◽

2002 ◽

Vol 30 (19) ◽

pp. 4314-4320 ◽

Cited By ~ 4

Author(s):

S.-W. Yang

Keyword(s):

Dna Polymerase ◽

Catalytic Properties ◽

Thermotoga Neapolitana ◽

Dna Polymerase I ◽

Nucleotide Addition ◽

Polymerase I

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Closing and opening of the RNA polymerase trigger loop

10.1101/817080 ◽

2019 ◽

Cited By ~ 1

Author(s):

Abhishek Mazumder ◽

Miaoxin Lin ◽

Achillefs N. Kapanidis ◽

Richard H. Ebright

Keyword(s):

Rna Polymerase ◽

Fluorescent Probe ◽

Active Center ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fluorescence ◽

Structural Element ◽

Trigger Loop ◽

Nucleotide Addition

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an “unfolded”/“open” state that allows an NTP substrate to enter the active center and a “folded”/“closed” state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

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Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

Genes & Development ◽

10.1101/gad.348874.121 ◽

2021 ◽

Author(s):

Lu Li ◽

Peike Sheng ◽

Tianqi Li ◽

Christopher J. Fields ◽

Nicholas M. Hiers ◽

...

Keyword(s):

Gene Expression ◽

Ubiquitin Ligase ◽

Transcriptional Repression ◽

Mirna Target ◽

Cross Linking ◽

Base Pairing ◽

Proapoptotic Protein ◽

Nucleotide Addition ◽

Mirna Degradation ◽

Target Rna

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA–target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3′ end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

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