Publications by authors named "Smita S Patel"

95 Publications

The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

EMBO J 2022 May 19;41(10):e109782. Epub 2022 Apr 19.

Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA.

The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.
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http://dx.doi.org/10.15252/embj.2021109782DOI Listing
May 2022

Assembly and Cryo-EM structure determination of yeast mitochondrial RNA polymerase initiation complex intermediates.

STAR Protoc 2021 06 31;2(2):100431. Epub 2021 Mar 31.

Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, KU Leuven, 3000 Leuven, Belgium.

In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of the component proteins and assembly of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1's structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results. For complete details on the use and execution of this protocol, please refer to De Wijngaert et al. (2021).
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http://dx.doi.org/10.1016/j.xpro.2021.100431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044712PMC
June 2021

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

Mol Cell 2021 01 4;81(2):268-280.e5. Epub 2020 Dec 4.

Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, KU Leuven, 3000 Leuven, Belgium. Electronic address:

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.
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http://dx.doi.org/10.1016/j.molcel.2020.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855493PMC
January 2021

Structure, mechanism, and regulation of mitochondrial DNA transcription initiation.

J Biol Chem 2020 12 30;295(52):18406-18425. Epub 2020 Oct 30.

Department of Biochemistry and Molecular Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, USA. Electronic address:

Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome (mitochondrial DNA) and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The re-constituted transcriptional complexes of (yeast) and humans, aided with high-resolution structures and biochemical characterizations, have provided a deeper understanding of the mechanism and regulation of mitochondrial DNA transcription. In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.
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http://dx.doi.org/10.1074/jbc.REV120.011202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939475PMC
December 2020

The dynamic landscape of transcription initiation in yeast mitochondria.

Nat Commun 2020 08 27;11(1):4281. Epub 2020 Aug 27.

School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.
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http://dx.doi.org/10.1038/s41467-020-17793-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452894PMC
August 2020

Phosphorylation of mitochondrial transcription factor B2 controls mitochondrial DNA binding and transcription.

Biochem Biophys Res Commun 2020 07 3;528(3):580-585. Epub 2020 Jun 3.

Hope College, Department of Chemistry, 35 E. 12th Street, Holland, MI, 49423, United States. Electronic address:

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial gene expression must be coordinated with nuclear gene expression to respond to cellular energetic needs. To gain insight into the coordination between the nucleus and mitochondria, there is a need to understand the regulation of transcription of mitochondrial DNA (mtDNA). Reversible protein post-translational modifications of the mtDNA transcriptional machinery may be one way to control mtDNA transcription. Here we focus on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, serine 197, and threonine 313. Phosphomimetics were established at these positions. Proteins were purified and analyzed for their ability to bind mtDNA and initiate transcription in vitro. Our results indicate phosphorylation at threonine 184 and threonine 313 impairs promoter binding and prevents transcription. These findings provide a potential regulatory mechanism of mtDNA transcription and help clarify the importance of protein post-translational modifications in mitochondrial function.
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http://dx.doi.org/10.1016/j.bbrc.2020.05.141DOI Listing
July 2020

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding.

J Biol Chem 2020 05 2;295(20):6823-6830. Epub 2020 Apr 2.

Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.
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http://dx.doi.org/10.1074/jbc.RA120.013338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242694PMC
May 2020

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

EMBO J 2020 03 9;39(6):e103367. Epub 2020 Feb 9.

Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA.

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.
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http://dx.doi.org/10.15252/embj.2019103367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073461PMC
March 2020

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Nucleic Acids Res 2020 03;48(5):2604-2620

Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.
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http://dx.doi.org/10.1093/nar/gkaa040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049685PMC
March 2020

Time-resolved analysis of transcription through chromatin.

Methods 2019 04 29;159-160:90-95. Epub 2019 Jan 29.

Fox Chase Cancer Center, Philadelphia, PA 19111, USA; Biology Faculty, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

During transcription along nucleosomal DNA, RNA polymerase II (Pol II) pauses at multiple positions and induces formation of multiple intermediates that aid in maintaining proper chromatin structure. To describe the kinetics of this multiple-step reaction, we utilized a computational model-based approach and KinTek Explorer software to analyze the time courses. Here we describe the stepwise protocol for analysis of the kinetics of transcription through a nucleosome that provides the rate constants for each step of this complex process. We also present an example where this time-resolved approach was applied to study the mechanism of histone chaperone FACT action during Pol II transcription through a single nucleosome by comparing the rate constants derived in the presence or in the absence of FACT.
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http://dx.doi.org/10.1016/j.ymeth.2019.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589111PMC
April 2019

HDX-MS reveals dysregulated checkpoints that compromise discrimination against self RNA during RIG-I mediated autoimmunity.

Nat Commun 2018 12 18;9(1):5366. Epub 2018 Dec 18.

Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, 33458, USA.

Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.
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http://dx.doi.org/10.1038/s41467-018-07780-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299088PMC
December 2018

Highly efficient 5' capping of mitochondrial RNA with NAD and NADH by yeast and human mitochondrial RNA polymerase.

Elife 2018 12 12;7. Epub 2018 Dec 12.

Department of Genetics and Waksman Institute, Rutgers University, United States.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD and NADH, using NAD and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD and NADH levels and adjusting transcriptional outputs accordingly.
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http://dx.doi.org/10.7554/eLife.42179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298784PMC
December 2018

RIG-I Uses an ATPase-Powered Translocation-Throttling Mechanism for Kinetic Proofreading of RNAs and Oligomerization.

Mol Cell 2018 10 27;72(2):355-368.e4. Epub 2018 Sep 27.

Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA. Electronic address:

RIG-I has a remarkable ability to specifically select viral 5'ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient-state kinetics, we elucidated the ATPase-driven "kinetic proofreading" mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off rates. ATP binding facilitates dsRNA engagement but, interestingly, makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merten syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5'ppp dsRNA but, more importantly, drives RIG-I oligomerization through translocation, which we show to be regulated by helicase motif IVa. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5'ppp end "throttles" translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.
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http://dx.doi.org/10.1016/j.molcel.2018.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434538PMC
October 2018

Helicase promotes replication re-initiation from an RNA transcript.

Nat Commun 2018 06 13;9(1):2306. Epub 2018 Jun 13.

Howard Hughes Medical Institute, Cornell University, Ithaca, NY, 14853, USA.

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.
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http://dx.doi.org/10.1038/s41467-018-04702-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997990PMC
June 2018

Correlating Transcription Initiation and Conformational Changes by a Single-Subunit RNA Polymerase with Near Base-Pair Resolution.

Mol Cell 2018 05;70(4):695-706.e5

Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Howard Hughes Medical Institute, Baltimore, MD 21205, USA; Departments of Biophysics and Biophysical Chemistry, Biophysics, and Biomedical Engineering, Johns Hopkins University, MD 21205, USA. Electronic address:

We provide a comprehensive analysis of transcription in real time by T7 RNA Polymerase (RNAP) using single-molecule fluorescence resonance energy transfer by monitoring the entire life history of transcription initiation, including stepwise RNA synthesis with near base-pair resolution, abortive cycling, and transition into elongation. Kinetically branching pathways were observed for abortive initiation with an RNAP either recycling on the same promoter or exchanging with another RNAP from solution. We detected fast and slow populations of RNAP in their transition into elongation, consistent with the efficient and delayed promoter release, respectively, observed in ensemble studies. Real-time monitoring of abortive cycling using three-probe analysis showed that the initiation events are stochastically branched into productive and failed transcription. The abortive products are generated primarily from initiation events that fail to progress to elongation, and a majority of the productive events transit to elongation without making abortive products.
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http://dx.doi.org/10.1016/j.molcel.2018.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983381PMC
May 2018

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases.

J Biol Chem 2017 11 7;292(44):18145-18160. Epub 2017 Sep 7.

From the Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School and

Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10), yeast mitochondrial Rpo41 (6 × 10), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10 The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine-purine mismatches were relatively frequent (10-10), whereas C→G, U→A, G→C, and C→A errors from purine-purine and pyrimidine-pyrimidine mismatches were rare (10-10). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of transcribed RNAs.
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http://dx.doi.org/10.1074/jbc.M117.797480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672038PMC
November 2017

Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation.

Nucleic Acids Res 2017 01 29;45(2):861-874. Epub 2016 Nov 29.

Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical school, Piscataway, NJ 08854, USA

Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has 'post-recruitment' roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from -4 to +1 in the open complex with all three proteins and from -4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar K values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery.
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http://dx.doi.org/10.1093/nar/gkw1157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314767PMC
January 2017

Overcoming a nucleosomal barrier to replication.

Sci Adv 2016 Nov 11;2(11):e1601865. Epub 2016 Nov 11.

Fox Chase Cancer Center, Philadelphia, PA 19111, USA.; Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia.

Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31-40) and +(41-65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop.
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http://dx.doi.org/10.1126/sciadv.1601865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5106197PMC
November 2016

DNA looping mediates nucleosome transfer.

Nat Commun 2016 11 3;7:13337. Epub 2016 Nov 3.

Department of Physics-Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853, USA.

Proper cell function requires preservation of the spatial organization of chromatin modifications. Maintenance of this epigenetic landscape necessitates the transfer of parental nucleosomes to newly replicated DNA, a process that is stringently regulated and intrinsically linked to replication fork dynamics. This creates a formidable setting from which to isolate the central mechanism of transfer. Here we utilized a minimal experimental system to track the fate of a single nucleosome following its displacement, and examined whether DNA mechanics itself, in the absence of any chaperones or assembly factors, may serve as a platform for the transfer process. We found that the nucleosome is passively transferred to available dsDNA as predicted by a simple physical model of DNA loop formation. These results demonstrate a fundamental role for DNA mechanics in mediating nucleosome transfer and preserving epigenetic integrity during replication.
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http://dx.doi.org/10.1038/ncomms13337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097161PMC
November 2016

The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

J Biol Chem 2016 08 16;291(32):16828-39. Epub 2016 Jun 16.

From the Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854,

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.
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http://dx.doi.org/10.1074/jbc.M116.740282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974394PMC
August 2016

Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork.

Methods 2016 10 9;108:65-78. Epub 2016 May 9.

Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical School, 683 Hoes Lane West, Piscataway 08854, NJ, USA. Electronic address:

Replicative helicases work closely with the replicative DNA polymerases to ensure that the genomic DNA is copied in a timely and error free manner. In the replisomes of prokaryotes, mitochondria, and eukaryotes, the helicase and DNA polymerase enzymes are functionally and physically coupled at the leading strand replication fork and rely on each other for optimal DNA strand separation and synthesis activities. In this review, we describe pre-steady state kinetic methods to quantify the base pair unwinding-synthesis rate constant, a fundamental parameter to understand how the helicase and polymerase help each other during leading strand replication. We describe a robust method to measure the chemical step size of the helicase-polymerase complex that determines how the two motors are energetically coupled while tracking along the DNA. The 2-aminopurine fluorescence-based method provide structural information on the leading strand helicase-polymerase complex, such as the distance between the two enzymes, their relative positions at the replication fork, and their roles in fork junction melting. The combined information garnered from these methods informs on the mutual dependencies between the helicase and DNA polymerase enzymes, their stepping mechanism, and their individual functions at the replication fork during leading strand replication.
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http://dx.doi.org/10.1016/j.ymeth.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035585PMC
October 2016

Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

Nucleic Acids Res 2016 05 16;44(9):4200-10. Epub 2016 Feb 16.

Rutgers University, Robert Wood Johnson Medical School, Department of Biochemistry and Molecular Biology, NJ 08854, USA

A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE-a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA.
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http://dx.doi.org/10.1093/nar/gkw098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872091PMC
May 2016

Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I.

Proc Natl Acad Sci U S A 2016 Jan 5;113(3):596-601. Epub 2016 Jan 5.

Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854

RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl (2'-OMe) group to the 5'-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5'ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2'-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2'-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution.
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http://dx.doi.org/10.1073/pnas.1515152113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725518PMC
January 2016

T7 replisome directly overcomes DNA damage.

Nat Commun 2015 Dec 17;6:10260. Epub 2015 Dec 17.

Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853, USA.

Cells and viruses possess several known 'restart' pathways to overcome lesions during DNA replication. However, these 'bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.
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http://dx.doi.org/10.1038/ncomms10260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703881PMC
December 2015

The autoinhibitory CARD2-Hel2i Interface of RIG-I governs RNA selection.

Nucleic Acids Res 2016 Jan 26;44(2):896-909. Epub 2015 Nov 26.

Robert Wood Johnson Medical School, Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, NJ 08854, USA

RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5' triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5'-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a 'gate' that prevents cellular RNAs from generating productive complexes that can signal.
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http://dx.doi.org/10.1093/nar/gkv1299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737149PMC
January 2016

Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

Nucleic Acids Res 2015 Jul 24;43(12):5924-35. Epub 2015 May 24.

Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus-Ter complex. The T7 replisome was arrested at the non-permissive end of Tus-Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus-Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.
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http://dx.doi.org/10.1093/nar/gkv527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4499146PMC
July 2015

Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other.

Elife 2015 May 13;4. Epub 2015 May 13.

Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, United States.

Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase (DNAP) enzymes, but the structural and mechanistic basis of coupling is poorly understood. This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities. Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting, but each enzyme melts the junction base pair partially. When combined, the junction base pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end. The synergistic shift in equilibrium of junction base pair melting by combined enzymes explains the cooperativity, wherein helicase stimulates the polymerase by promoting dNTP binding (decreasing dNTP Km), polymerase stimulates the helicase by increasing the unwinding rate-constant (kcat), consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA.
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http://dx.doi.org/10.7554/eLife.06562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460406PMC
May 2015

Finding the right match fast.

Cell 2015 Feb;160(5):809-811

Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, 675 Hoes Lane West, Piscataway, NJ 08854, USA. Electronic address:

DNA recombinases face the daunting task of locating and pairing up specific sequences among millions of base pairs in a genome, all within about an hour. Qi et al. show that recombinases solve this problem by searching in 8-nt microhomology units, reducing the search space and accelerating the homology search.
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http://dx.doi.org/10.1016/j.cell.2015.02.007DOI Listing
February 2015

Interactions of the yeast mitochondrial RNA polymerase with the +1 and +2 promoter bases dictate transcription initiation efficiency.

Nucleic Acids Res 2014 Oct 23;42(18):11721-32. Epub 2014 Sep 23.

Department of Biochemistry and Molecular Biology, RUTGERS-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA

Mitochondrial promoters of Saccharomyces cerevisiae share a conserved -8 to +1 sequence with +1+2 AA, AG or AT initiation sequence, which dictates the efficiency of transcription initiation by the mitochondrial RNA polymerase Rpo41 and its initiation factor Mtf1. We used 2-aminopurine fluorescence to monitor promoter melting and measured the kcat/Km of 2-mer synthesis to quantify initiation efficiency with systematic changes of the +1+2 base pairs to matched and mismatched pairs. We show that AA promoters are most efficient, followed by AG and then AT promoters, and the differences in their efficiencies stem specifically from differential melting of +1+2 region without affecting melting of the upstream -4 to -1 region. Inefficient +1+2 melting increases the initial NTPs Kms of the AG and AT promoters relative to AA or singly mispaired promoters. The 16-100-fold higher catalytic efficiency of AA initiation sequence relative to AG and AT, respectively, is partly due to Rpo41-Mtf1 interactions with the +1+2 non-template adenines that generate a stable pre-transcribing complex. We propose a model where the +2 base pair regulates the efficiency of initial transcription by controlling multiple steps including downstream promoter opening, +1+2 NTPs binding, and the rate of 2-mer synthesis.
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http://dx.doi.org/10.1093/nar/gku868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191429PMC
October 2014

Fluorescent methods to study transcription initiation and transition into elongation.

Exp Suppl 2014;105:105-30

Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.
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http://dx.doi.org/10.1007/978-3-0348-0856-9_6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430081PMC
October 2014
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