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PMID: 15100439 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants.

RNA (New York, N.Y.) ·Vol. 10 ·No. 5 ·2004-05-00 ·Pages 841-53

Sissler M, Helm M, Frugier M, Giege R, Florentz C

Abstract

In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C.A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.

MeSH Terms
Acylation Aminoacyltransferases/genetics Genetic Variation Humans MERRF Syndrome/genetics Mitochondria/genetics Mitochondrial Diseases/genetics Mutation Nucleic Acid Conformation RNA, Transfer, Lys/genetics Sequence Analysis, RNA
Chemicals
RNA, Transfer, Lys Aminoacyltransferases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Sissler Marie
UPR 9002 du CNRS, Département Mécanismes et Macromolécules de la Synthèse Protéique, et Cristallogenèse, Institut de Biologie Moléculaire et Cellulaire, F-67084 Strasbourg Cedex, France.
Helm Mark
Frugier Magali
Giege Richard
Florentz Catherine
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1261/rna.5267604
Published
2004-05-00
Pages
841-53
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1370574
Subset
IM
External Links
PubMed source DOI: 10.1261/rna.5267604 PMC Full Text View journal details
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