Cloning and Expression of Mitochondrial Translational Elongation Factor Ts from Bovine and Human Liver

Hong Xin,Velinda Woriax,William Burkhart,Linda L Spremulli

doi:10.1074/jbc.270.29.17243

Abstract

The sequences of the cDNAs for the mitochondrial translational elongation factor Ts (EF-Tsmt) from bovine and human liver have been obtained. The deduced amino acid sequence of bovine liver EF-Tsmt is 338 residues in length and includes a 55-amino acid signal peptide and a mature protein of 283 residues. The sequence of the mature form of bovine EF-Tsmt is 91% identical to that of human EF-Tsmt and 29% identical to Escherichia coli EF-Ts. Southern analysis indicates that there are two genes for EF-Tsmt in bovine liver chromosomal DNA. A 224-base pair intron is located near the 5'-end of at least one of these genes. Northern analysis using a human multiple tissue blot indicates that EF-Tsmt is expressed in all tissues, with the highest levels of expression in skeletal muscle, liver, and kidney. Both the mature and precursor forms of bovine liver EF-Tsmt have been expressed in E. coli as histidine-tagged proteins. The mature form of EF-Tsmt forms a complex with E. coli elongation factor Tu. This complex is active in poly(U)-directed polymerization of phenylalanine. The precursor form is expressed as a 42-kDa protein, which is rapidly degraded in the cell.

Highlights

Cloning of Bovine and Human Liver EF-Tsm t cDNAs-EFTSrn t is the product of a nuclear gene in mammals
The EF-Tu·Tsrn t complex was dissociated, and the two factors were separated by reversephase high performance liquid chromatography
EF-Tsrn t was subjected to NH2-terminal Edman degradation and to internal peptide sequence analysis following digestion with endoproteinase Lys-C

Summary

To whom correspondence should be addressed

Ciated in the absence of protein-denaturing reagents [4]. In this organism, GDP present in the EF-Tu'GDP complex is thought to exchange directly with GTP present in the The EFTU'Tsmt complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides In this respect, the mitochondrial factors differ significantly from the corresponding E. coli factors and show some resemblance to thermophilic EF-Tu and EF-Ts. The stability of the EF-Tu'EF-Ts complex is thought to be determined largely by the nature of the EF-Ts component. Chloroplast EF-Ts from Euglena gracilis forms a tighter complex with E. coli and chloroplast EF-Tu than does the E. coli factor [8] It is not clear what features ofEF-Ts modulate the strength of its interaction with EF-Tu. The genes for EF-Ts from several prokaryotes have been cloned and sequenced, as has the gene for chloroplast EF-Ts from the thermophilic red algae Galdieria sulphuraria. Bovine liver EF-Tsmt has been expressed in E. coli

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

F E CGE GEDAADAE