Sequence and Identiflcation of the Nucieotide Binding Site for the Elongation Factor Tu from Thermits Thermophtius Hb8

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T.thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T.thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain. INTRODUCTION Elongation factor Tu (EF-Tu) from E .coli mediates the binding of aminoacyl-tRNA to programmed ribosomes (1) and plays a role in maintaining the fidelity of translation (2) . Amino acid sequences were determined for the procaryotic elongation factors from E. coli (3, 4), yeast mitochondrium (5) chloroplasts of Euqlena qracilis (6) as well as for several eucaryotic elongation factor I a species (7, 8, 9). EF-Tu possesses some sequence homologies with eucaryotic elongation factor la and G-proteins mainly in the guanosin-nucleotide binding domain (10). EF-Tu from T.thermophilus HB8 is temperature insensitive up to 65 °C (11) in contrast to EF-Tu from E.coli. It forms stable aa-tRNA»EF-Tu>GTP ternary complexes and is well suited for physical and biochemical investigations. We used this thermophilic EFTu to construct affinity columns for the isolation of aminoacyltRNAs (12) and for the investigation of aminoacyl-tRNA • EF-Tu*GTP interactions (13). In order to prepare sufficient quantities of the thermophilic elongation factor and its mutants for physical © IR L Press Limited, Oxford, England. 9 2 6 3 Nucleic Acids Research studies the expression of its genes in E.coli was attempted. In this communication we present the sequence of one of the two T.thermophilus EF-Tu genes and the identification, by affinity labeling, of the nucleotide binding site in the protein. MATERIALS AND METHODS Enzymes, deoxynucleoside-5'-triphosphates and dideoxynucleosides-5'-triphosphates were obtained from Pharmacia (Uppsala, Sweden), BRL (Eggenstein, FRG) and Boehringer (Mannheim, FRG). [H]GDP (11.3 Ci/mmole), [UC]GTP (500 mCi/mmole), S-labeled methionine (1275 Ci/mmole) and 'Amplify' were purchased from Amersham-Buchler (Braunschweig, FRG). [ P ]orthophosphate and P-labeled dATP (800 Ci/ mmole) were obtained from Du Pont/New England Nuclear (Bad Nauheim, FRG). [GP]GDP (1000 Ci/mmole) was prepared as described elsewhere (14). CM-Sepharose CL-6B and Sephadex LH-60 were from Pharmacia. NalO., NaBH3(CN) and NaBH. were from Serva (Heidelberg, FRG). Acrylamide and N,N'methylenebisacrylamide were obtained from BRL. All other reagents were of analytical grade and purchased from Merck (Darmstadt, FRG). The plasmid pPR 1 carrying the 1.0 kbp Nrul/Hpal fragment of the E.coli tufA gene was obtained from Dr. L. Bosch (Leiden). E.coli strains RR1 and Kill were U8ed for cloning experiments. The E.coli strain R312A which was used for minicell expression was from Dr. N. Schumann (Bayreuth). For hybridization experiments 20 p.g of T•thermophilus chromosomal DNA were digested with several restriction enzymes using 33 mM Tri8-acetate pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, 0.1 mg/ml BSA as incubation buffer, in a final volume of 60 jil. Cleavage was complete after 3 h at 37 C in the presence of 20 units of restriction enzyme. The resulting DNA fragments were separated by horizontal gel electrophoresis (1.3 % agarose in 40 mM Tris-acetate pH 7.9, 25 mM sodium acetate, 0.5 mM EDTA) run at 3 V/cm for 4 h. Transfer of separated 32 DNA to a nitrocellulose filter and hybridization to the Plabeled E.coli tufA fragment of pPR 1 were carried out as published elsewhere (15, 16).