Linked Life Data

10390340

Source:http://linkedlifedata.com/resource/pubmed/id/10390340

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1999-7-30
pubmed:abstractText
The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNAAlais critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNAAla. In doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNAAlawith respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNAAla. A key observation is that the extent of aminoacylation of tRNAAlawas independent of tRNAAlaconcentration over a 75-fold range. Therefore, the cellular assay of tRNAAlareflects the substrate quality of the molecule for formation of alanyl-tRNAAla. These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0022-2836
pubmed:author
pubmed:copyrightInfo
Copyright 1999 Academic Press.
pubmed:issnType
Print
pubmed:day
9
pubmed:volume
290
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
391-409
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:10390340-Acylation, pubmed-meshheading:10390340-Alanine-tRNA Ligase, pubmed-meshheading:10390340-Base Sequence, pubmed-meshheading:10390340-Blotting, Northern, pubmed-meshheading:10390340-Escherichia coli, pubmed-meshheading:10390340-Genes, Suppressor, pubmed-meshheading:10390340-Guanosine Triphosphate, pubmed-meshheading:10390340-Lysine, pubmed-meshheading:10390340-Mutation, pubmed-meshheading:10390340-Peptide Elongation Factor Tu, pubmed-meshheading:10390340-Protein Binding, pubmed-meshheading:10390340-RNA, Bacterial, pubmed-meshheading:10390340-RNA, Transfer, Ala, pubmed-meshheading:10390340-RNA, Transfer, Amino Acyl, pubmed-meshheading:10390340-Reproducibility of Results, pubmed-meshheading:10390340-Structure-Activity Relationship, pubmed-meshheading:10390340-Suppression, Genetic
pubmed:year
1999
pubmed:articleTitle
The reliability of in vivo structure-function analysis of tRNA aminoacylation.
pubmed:affiliation
Department of Bacteriology, University of Wisconsin, Madison, WI, 53706-1567, USA. wmcclain@facstaff.wisc.edu
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.