Structure-guided mutational analysis of T4 RNA ligase 1.

Structure-guided mutational analysis of T4 RNA ligase 1. Research Abstract Details 

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Structure-guided mutational analysis of T4 RNA ligase 1. Abstract Text:

Li Kai Wang,Beate Schwer,Stewart Shuman,

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.

Structure-guided mutational analysis of T4 RNA ligase 1. Publishing Authors By Initials

LK Wang,B Schwer,S Shuman,

For similar proteins: viral proteins research abstracts see: proteins: viral proteins research

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MEDLINE DATE:

Structure-guided mutational analysis of T4 RNA ligase 1. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: RNA (New York, N.Y.)

VOLUME: 12

Page Numbers: 2126-34

Journal Abbreviation: RNA

ISSN: 1355-8382

DAY: 26

MONTH: 10

YEAR: 2006

Structure-guided mutational analysis of T4 RNA ligase 1. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 9509184

Structure-guided mutational analysis of T4 RNA ligase 1. Keywords Mesh Terms:

KEYWORDS: Viral Proteins

MESH TERMS: metabolism

Chemical & Substance for Abstract: Structure-guided mutational analysis of T4 RNA ligase 1. Information

Substance Name: bacteriophage T4 RNA ligase 1

Registry Number: EC 6.5.1.3

Grant and Affiliation Information for Structure-guided mutational analysis of T4 RNA ligase 1.

AFFILIATION: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM42498

ACRONYM: GM

MEDLINETA: RNA

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