PMID:8323579
| Citation |
Garcia, GA, Koch, KA and Chong, S (1993) tRNA-guanine transglycosylase from Escherichia coli. Overexpression, purification and quaternary structure. J. Mol. Biol. 231:489-97 |
|---|---|
| Abstract |
tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the posttranscriptional modification of specific tRNAs (Asn, Asp, His and Tyr) with queuine. In E. coli this modification occurs via a two-step reaction: (1) TGT-catalyzed base exchange of guanosine-34 with preQ1 (7-aminomethyl-7-deazaguanine) and (2) addition of a cyclopentenediol moiety to the preQ1-34 tRNA. E. coli TGT is normally expressed at very low levels (approximately 1 mg from 500 g cells). The sequence of the queuine operon of E. coli has recently been reported by Reuter et al. (1991). We have cloned the tgt gene into an overexpressing vector in order to provide a more efficient preparation of TGT. A simple, four-step purification scheme yields 78 mg of homogeneous TGT per liter of cell culture (A600 = 5 to 6). Amino-terminal protein sequencing confirms the identity of the recombinant protein and indicates that the initiator methionine is retained in the mature form. Native-PAGE of TGT and SDS-PAGE of cross-linked TGT are most consistent with a hexameric quaternary structure for the enzyme. The cross-linking data also suggests that the enzyme exists as a dimer of trimers of identical 42.5 kDa subunits (total M(r) = 255 kDa. The enzyme is inactivated by cross-linking with the bisimidoester, dimethylsuberimidate. Substrate (tRNA) protects the enzyme against cross-linking and inactivation by dimethylsuberimidate and against inactivation by modification with ethylacetimidate, a monofunctional, imidoester. This indicates that the enzymic residues (presumably lysines) that are involved in cross-linking and the inactivation are in the active site of the enzyme. |
| Links |
PubMed Online version:10.1006/jmbi.1993.1296 |
| Keywords |
Amino Acids/analysis; Base Sequence; Cloning, Molecular; Cross-Linking Reagents; Dimethyl Suberimidate; Escherichia coli/enzymology; Guanine/analogs & derivatives; Guanine/metabolism; Molecular Sequence Data; Pentosyltransferases/biosynthesis; Pentosyltransferases/chemistry; Pentosyltransferases/genetics; Protein Conformation; RNA, Messenger/genetics; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Sequence Analysis; Transcription, Genetic |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.
