Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF-Tu and BstG-domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF-Tu. In contrast, the EcG-domain bound the nucleotides with much lower, micromolar affinities. The exchange of domains 2 and 3 had essentially no effect on the GDP-binding activity; all complexes of chimeric EF-Tus with GDP retained K(d) values in the nanomolar range. The final thermostability level of either EF-Tu was the result of a cooperative interaction between the G-domains and domains 2 + 3. The G-domains set up a "basic" level of the thermostability, which was approximately 20 degrees C higher with the BstG-domain than with the EcG-domain. This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG-domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent-accessible surface area. Domains 2 + 3 contributed by further stabilization of alpha-helical regions and, in turn, the functions of the G-domains to the level of the respective growth temperature optima. Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state. It was lower in the GTP conformation, and the mechanism involved the destabilization of the alpha-helical regions of the G-domain by Ecdomain 2.

• MeSH
• Amino Acids chemistry   MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Circular Dichroism MeSH
• Protein Denaturation MeSH
• Species Specificity MeSH
• Peptide Elongation Factor Tu chemistry   genetics   metabolism   MeSH
• Escherichia coli chemistry   MeSH
• Genetic Variation MeSH
• Geobacillus stearothermophilus chemistry   MeSH
• Guanosine Diphosphate metabolism   MeSH
• Guanosine Triphosphate metabolism   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Escherichia coli Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Fusion Proteins chemistry   isolation & purification   metabolism   MeSH
• Protein Folding MeSH
• Protein Structure, Secondary MeSH
• Temperature MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Amino Acids MeSH
• Bacterial Proteins MeSH
• Peptide Elongation Factor Tu MeSH
• Guanosine Diphosphate MeSH
• Guanosine Triphosphate MeSH
• Escherichia coli Proteins MeSH
• Recombinant Fusion Proteins MeSH

The complete primary structure of the str operon of Bacillus stearothermophilus was determined. It was established that the operon is a five-gene transcriptional unit: 5'-ybxF (unknown function; homology to eukaryotic ribosomal protein L30)-rpsL (S12)-rpsG (S7)-fus (elongation factor G [EF-G])-tuf (elongation factor Tu [EF-Tu])-3'. The main operon promoter (strp) was mapped upstream of ybxF, and its strength was compared with the strength of the tuf-specific promoter (tufp) located in the fus-tuf intergenic region. The strength of the tufp region to initiate transcription is about 20-fold higher than that of the strp region, as determined in chloramphenicol acetyltransferase assays. Deletion mapping experiments revealed that the different strengths of the promoters are the consequence of a combined effect of oppositely acting cis elements, identified upstream of strp (an inhibitory region) and tufp (a stimulatory A/T-rich block). Our results suggest that the oppositely adjusted core promoters significantly contribute to the differential expression of the str operon genes, as monitored by the expression of EF-Tu and EF-G.

• MeSH
• Genes, Bacterial * MeSH
• Peptide Elongation Factor G metabolism   MeSH
• Peptide Elongation Factor Tu biosynthesis   MeSH
• Genetic Vectors MeSH
• Geobacillus stearothermophilus genetics   MeSH
• Cloning, Molecular MeSH
• Molecular Sequence Data MeSH
• Operon MeSH
• Promoter Regions, Genetic MeSH
• Genes, Regulator MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Peptide Elongation Factor G MeSH
• Peptide Elongation Factor Tu MeSH