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
• aminokyseliny chemie   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• cirkulární dichroismus MeSH
• denaturace proteinů MeSH
• druhová specificita MeSH
• elongační faktor Tu chemie   genetika   metabolismus   MeSH
• Escherichia coli chemie   MeSH
• genetická variace MeSH
• Geobacillus stearothermophilus chemie   MeSH
• guanosindifosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• teplota MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aminokyseliny MeSH
• bakteriální proteiny MeSH
• elongační faktor Tu MeSH
• guanosindifosfát MeSH
• guanosintrifosfát MeSH
• proteiny z Escherichia coli MeSH
• rekombinantní fúzní proteiny 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
• bakteriální geny * MeSH
• elongační faktor G metabolismus   MeSH
• elongační faktor Tu biosyntéza   MeSH
• genetické vektory MeSH
• Geobacillus stearothermophilus genetika   MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• operon MeSH
• promotorové oblasti (genetika) MeSH
• regulační geny MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elongační faktor G MeSH
• elongační faktor Tu MeSH