Linked Life Data

12549933

Source:http://linkedlifedata.com/resource/pubmed/id/12549933

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2003-1-28
pubmed:abstractText
Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/2-thiouridine, http://linkedlifedata.com/resource/pubmed/chemical/5-methylaminomethyl-2-thiouridine, http://linkedlifedata.com/resource/pubmed/chemical/Aminopeptidases, http://linkedlifedata.com/resource/pubmed/chemical/Anticodon, http://linkedlifedata.com/resource/pubmed/chemical/Carbon-Sulfur Lyases, http://linkedlifedata.com/resource/pubmed/chemical/Escherichia coli Proteins, http://linkedlifedata.com/resource/pubmed/chemical/RNA, Transfer, Amino Acyl, http://linkedlifedata.com/resource/pubmed/chemical/RNA-Binding Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Sulfides, http://linkedlifedata.com/resource/pubmed/chemical/Sulfur, http://linkedlifedata.com/resource/pubmed/chemical/Thiouridine, http://linkedlifedata.com/resource/pubmed/chemical/cysteine desulfurase, http://linkedlifedata.com/resource/pubmed/chemical/peptidase T, http://linkedlifedata.com/resource/pubmed/chemical/persulfides, http://linkedlifedata.com/resource/pubmed/chemical/tRNA, glutamic acid-, http://linkedlifedata.com/resource/pubmed/chemical/tRNA, lysine-
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
4
pubmed:volume
42
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1109-17
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli.
pubmed:affiliation
School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, Wisconsin 53706, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't