Kinetic Isotope Effect Analysis of RNA 2'-O-Transphosphorylation.

Abstract:

:The breaking of RNA strands by 2'-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2'-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending on how the reaction is catalyzed. Because of this complexity and the lack of experimental information on transition-state structure, pinning down the chemical details of enzyme-catalyzed RNA strand cleavage has been difficult. Kinetic isotope effects (KIEs) provide information about changes in bonding as a reaction proceeds from ground state to transition state, and therefore they provide a powerful tool for revealing mechanistic detail. Application of kinetic isotope analyses to RNA 2'-O-transphosphorylation faces three fundamental challenges: synthesis of RNA substrate isotopomers with 18O substitutions at the 2'-O, 5'-O and nonbridging phosphoryl oxygens; determination of the 18O/16O ratios in the residual unreacted substrate or product RNAs; and analyzing these data to allow calculation of the KIEs for use in evaluating different mechanistic scenarios. In this chapter, we outline methods for surmounting these challenges for solution RNA 2'-O-transphosphorylation reactions, and we describe their initial application to understand nonenzymatic solution reactions and reactions catalyzed by the enzyme ribonuclease A.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Harris ME,York DM,Piccirilli JA,Anderson VE

doi

10.1016/bs.mie.2017.07.017

subject

Has Abstract

pub_date

2017-01-01 00:00:00

pages

433-457

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(17)30247-1

journal_volume

596

pub_type

杂志文章
  • Screening and characterizing human NAT2 variants.

    abstract::Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)00011-X

    authors: Savulescu MR,Mushtaq A,Josephy PD

    更新日期:2005-01-01 00:00:00

  • T cell receptor engineering.

    abstract::T lymphocytes express on their surface a heterodimeric αβ receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibod...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-396962-0.00008-2

    authors: Stone JD,Chervin AS,Aggen DH,Kranz DM

    更新日期:2012-01-01 00:00:00

  • Interactions between endosomal maturation and autophagy: analysis of ESCRT machinery during Caenorhabditis elegans development.

    abstract::Endocytosis and autophagy are key vesicular pathways involved in degradation and recycling of cellular material. Both degradative pathways finally fuse with lysosome but are indeed interconnected at several levels. In particular, the fusion between endosomes and autophagosomes can generate intermediate vesicles named ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-397926-1.00006-8

    authors: Manil-Ségalen M,Culetto E,Legouis R,Lefebvre C

    更新日期:2014-01-01 00:00:00

  • Near-infrared photoactivatable nitric oxide donors with photoacoustic readout.

    abstract::In this chapter, we motivate the need for photoactivatable NO donor molecules and give a brief survey of the existing chemical tools in the field. We then provide detailed protocols for the synthesis and validation of a near-infrared light-activated NO donor molecule, photoNOD-1, developed in our research group. With ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.05.003

    authors: Zhou EY,Knox HJ,Reinhardt CJ,Partipilo G,Chan J

    更新日期:2020-01-01 00:00:00

  • Assays for ubiquitin-like protein ligation and proteasome function in archaea.

    abstract::Ubiquitin-like protein (Ubl) ligation is common to diverse archaea and targets many cellular pathways, including those associated with sulfur mobilization, and also tags proteins as substrates for degradation by the proteasome. Here we highlight protocols to assay proteasome function and Ubl ligation in archaea. A cha...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.12.036

    authors: Fu X,Adams Z,Maupin-Furlow J

    更新日期:2019-01-01 00:00:00

  • Solving serpin crystal structures.

    abstract::Essentially the same steps are required to solve the crystal structure of a serpin as for any other protein: produce and purify protein, grow crystals, collect diffraction data, find estimates of the phase angles, and then refine and validate the structure. For the phasing step, experimental phasing methods involving ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385950-1.00004-3

    authors: Read RJ,Zhou A,Stein PE

    更新日期:2011-01-01 00:00:00

  • Glycoprotein maturation and the UPR.

    abstract::Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycos...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385928-0.00010-9

    authors: Hülsmeier AJ,Welti M,Hennet T

    更新日期:2011-01-01 00:00:00

  • Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems.

    abstract::Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been stu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)08012-8

    authors: Reardon JT,Sancar A

    更新日期:2006-01-01 00:00:00

  • Cytotoxic and cytoprotective actions of O2- and NO (ONOO-) are determined both by cellular GSH level and HO activity in macrophages.

    abstract::Survival of macrophages, which serve as the first-line defense against invading pathogens by invoking the overproduction of highly toxic peroxynitrite (ONOO-), depends on their ability to maintain the intracellular GSH level and to induce the expression of heme oxygenase-1 (HO-1). The ONOO- is produced by macrophages ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)96035-7

    authors: Srisook K,Kim C,Cha YN

    更新日期:2005-01-01 00:00:00

  • Precisely and Accurately Inferring Single-Molecule Rate Constants.

    abstract::The kinetics of biomolecular systems can be quantified by calculating the stochastic rate constants that govern the biomolecular state vs time trajectories (i.e., state trajectories) of individual biomolecules. To do so, the experimental signal vs time trajectories (i.e., signal trajectories) obtained from observing i...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.08.021

    authors: Kinz-Thompson CD,Bailey NA,Gonzalez RL Jr

    更新日期:2016-01-01 00:00:00

  • Establishing the Architecture of Plant Gene Regulatory Networks.

    abstract::Gene regulatory grids (GRGs) encompass the space of all the possible transcription factor (TF)-target gene interactions that regulate gene expression, with gene regulatory networks (GRNs) representing a temporal and spatial manifestation of a portion of the GRG, essential for the specification of gene expression. Thus...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.03.003

    authors: Yang F,Ouma WZ,Li W,Doseff AI,Grotewold E

    更新日期:2016-01-01 00:00:00

  • Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.

    abstract::It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.01.012

    authors: Lin Y,May GE,Joel McManus C

    更新日期:2015-01-01 00:00:00

  • Quantitative assays of Mdm2 ubiquitin ligase activity and other ubiquitin-utilizing enzymes for inhibitor discovery.

    abstract::Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor ac...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)99046-0

    authors: Auger KR,Copeland RA,Lai Z

    更新日期:2005-01-01 00:00:00

  • TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

    abstract::Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1. ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.04.024

    authors: Young AP,Bandarian V

    更新日期:2018-01-01 00:00:00

  • Stabilized plasmid-lipid particles: a systemic gene therapy vector.

    abstract::The ability of a systemically administered gene therapy vector to exhibit extended circulation lifetimes, accumulate at a distal tumor site, and enable transgene expression is unique to SPLP. The flexibility and low toxicity of SPLP as a platform technology for systemic gene therapy allows for further optimization of ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(02)46048-x

    authors: Fenske DB,MacLachlan I,Cullis PR

    更新日期:2002-01-01 00:00:00

  • Differential display analysis of gene expression altered by ras oncogene.

    abstract::The goal of the signal transduction pathways, such as those controlled by Ras, is in large part to ensure highly stringent regulation of the target genes in the nucleus, which are collectively responsible for the signal output, or phenotypes, of the cell. Understanding of the Ras effect ultimately requires the identif...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(01)32206-1

    authors: Jo H,Cho YJ,Zhang H,Liang P

    更新日期:2001-01-01 00:00:00

  • Nuclear magnetic resonance measurements of slow conformational dynamics in macromolecules.

    abstract::The combined use of rotating-frame relaxation methods, temperature-dependent measurements of line shapes and magnetization transfer experiments allows in favorable cases the examination in some detail of exchange processes that occur on the millisecond time scale. It is possible to determine not only the rate constant...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/s0076-6879(94)39023-1

    authors: Lane AN,Lefèvre JF

    更新日期:1994-01-01 00:00:00

  • Roles of GlcNAc-6-O-sulfotransferases in lymphoid and nonlymphoid tissues.

    abstract::Recent studies using sulfotransferase-deficient mice have revealed various physiological functions of sulfated glycans. Studies using gene-targeted mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 showed that these sulfotransferases play critical roles in lymphocyte homing....

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(10)79014-5

    authors: Kawashima H

    更新日期:2010-01-01 00:00:00

  • Sensitive detection of structural features and rearrangements in long, structured RNA molecules.

    abstract::Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suite...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.04.002

    authors: Adams RL,Huston NC,Tavares RCA,Pyle AM

    更新日期:2019-01-01 00:00:00

  • Reannotation of Genomes by Means of Proteomics Data.

    abstract::Omics approaches have become popular in biology as powerful discovery tools, and currently gain in interest for diagnostic applications. Establishing the accurate genome sequence of any organism is easy, but the outcome of its annotation by means of automatic pipelines remains imprecise. Some protein-encoding genes ma...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.09.019

    authors: Armengaud J

    更新日期:2017-01-01 00:00:00

  • Use of phosphorodithioate-based compounds as hydrogen sulfide donors.

    abstract::Synthetic hydrogen sulfide (H2S) donors are useful research tools as well as potential therapeutic agents. In this chapter, we report the detailed protocols for the synthesis and evaluation of a series of phosphorodithioate-based H2S donors. Fluorescence assays were used to determine H2S release from the donors in bot...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2014.11.032

    authors: Park CM,Xian M

    更新日期:2015-01-01 00:00:00

  • Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy.

    abstract::We have described procedures for collecting, processing, and analyzing kinetic data obtained by photobleaching microscopy of GFP-tagged chromatin proteins in nuclei of cultured living cells. These procedures are useful for characterizing the in vivo binding of chromatin proteins to their natural template--unperturbed,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(03)75025-3

    authors: Phair RD,Gorski SA,Misteli T

    更新日期:2004-01-01 00:00:00

  • Molecular tools for investigating ANME community structure and function.

    abstract::Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabol...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385112-3.00004-4

    authors: Hallam SJ,Pagé AP,Constan L,Song YC,Norbeck AD,Brewer H,Pasa-Tolic L

    更新日期:2011-01-01 00:00:00

  • Analysis of posttranslational regulations in the Neurospora circadian clock.

    abstract::Posttranslational modification of circadian clock proteins by phosphorylation is an essential regulatory process in the control of eukaryotic circadian clocks. In the Neurospora circadian clock, the key clock protein FREQUENCY (FRQ) is progressively phosphorylated. The phosphorylation of FRQ is regulated by both kinas...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)93017-6

    authors: Liu Y

    更新日期:2005-01-01 00:00:00

  • Creating recombination-activated genes and sequence-defined mutant libraries using transposons.

    abstract::The properties of a collection of transposon Tn5 derivatives that generate reporter gene fusions and internal protein tags are summarized. Procedures utilizing several of the transposons for generating genes activated by Cre-loxP recombination and for creating large sequence-defined mutant libraries are described in d...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)21012-7

    authors: Gallagher L,Turner C,Ramage E,Manoil C

    更新日期:2007-01-01 00:00:00

  • Purification and reconstitution of PYP-phytochrome with biliverdin and 4-hydroxycinnamic acid.

    abstract::PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoic...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)22009-3

    authors: Chung YH,Masuda S,Bauer CE

    更新日期:2007-01-01 00:00:00

  • Analysis of interactions between intraflagellar transport proteins.

    abstract::Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii, IFT A contains 6 d...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-397945-2.00010-X

    authors: Behal RH,Cole DG

    更新日期:2013-01-01 00:00:00

  • Transport of a caspase inhibitor across the blood-brain barrier by chitosan nanoparticles.

    abstract::The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Severa...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-391860-4.00013-6

    authors: Yemişci M,Gürsoy-Özdemir Y,Caban S,Bodur E,Capan Y,Dalkara T

    更新日期:2012-01-01 00:00:00

  • Type I polyketide synthases that require discrete acyltransferases.

    abstract::The diverse structures of polyketide natural products are reflected by the equally diverse polyketide biosynthetic enzymes, namely polyketide synthases (PKSs). Three major classes of PKSs are known-noniterative type I PKSs, iterative type II PKSs and acyl carrier protein-independent type III PKSs, each of which consis...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)04608-4

    authors: Cheng YQ,Coughlin JM,Lim SK,Shen B

    更新日期:2009-01-01 00:00:00

  • Measurement of inverse agonism in β-adrenoceptors.

    abstract::Increasing numbers of compounds, previously classified as antagonists, were shown to inhibit this spontaneous or constitutive receptor activity, instead of leave it unaffected as expected for a formal antagonist. In addition, some other antagonists did not have any effect by themselves, but prevented the inhibition of...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-381296-4.00003-8

    authors: Taira CA,Monczor F,Höcht C

    更新日期:2010-01-01 00:00:00