Manipulation of the tuf gene provides clues to the localization of sequence element(s) involved in the thermal stability of Thermotoga maritima elongation factor Tu.
Truncated versions of the tuf gene for elongation factor Tu (EF-Tu; 400 aa) from the hyperthermophilic bacterium Thermotoga maritima have been produced by progressive 3'-->5' trimming. The truncated genes have been expressed in Escherichia coli and the thermal stability of the gene products has been assayed by monitoring their GDP-binding capacity after preheating the cell-free extracts at various temperatures (65-95 degrees C). One of the truncated proteins, corresponding to the nucleotide-binding domain (G domain; aa 1-200) appears to be only slightly less stable than the full-length EF-Tu. Replacement of the first 90 N-terminal residues of both the full-length Thermotoga EF-Tu and the isolated G domain with the corresponding sequence of the mesophilic bacterium E. coli, drastically destabilizes both the complete and the truncated protein, indicating that sequence element(s) that are crucial for the attainment of a thermally stable conformation of the Thermotoga EF-Tu lie well within the initial portion of the G domain between residues 1 and 90. The relevant residues defy identification, however, as no amino acid preferences, or exclusive sequence element(s), appear to distinguish the N-terminal region of the thermophilic proteins from those of mesophilic counterparts. It is suggested that the thermal stability of Thermotoga EF-Tu is critically dependent upon unique tertiary structural interactions involving certain N-terminal residues of the molecule.
Sanangelantoni
A M
AM
Dipartimento di Genetica e Microbiologia A. Buzzati Traverso, Università di Pavia, Italy. Sanan@ipvgen.unipv.it
Cammarano
P
P
Tiboni
O
O
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology; D002678:Chimera; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D010957:Plasmids; D011485:Protein Binding; D011995:Recombination, Genetic; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D006153:Guanosine Diphosphate; D010444:Peptide Elongation Factor Tu