Genetic engineering, isolation and characterization of a truncated Escherichia coli elongation factor Tu comprising domains 2 and 3.
A deletion mutant of a plasmid born Escherichia coli tufA gene, which codes for a truncated elongation factor Tu comprising domains 2 and 3, has been constructed by genetic engineering. This gene was overexpressed in E. coli, and a polypeptide representing the truncated elongation factor Tu was isolated, purified to near homogeneity, crystallized and characterized physico-chemically as well as biochemically. Circular dichroism spectroscopy and limited tryptic digestion demonstrate that the isolated domain pair 2 and 3 behaves like an independent folding unit which adopts a similar secondary and most likely, tertiary, structure to that present in the intact elongation factor Tu. However, the isolated domain pair 2 and 3 does not interact with aminoacyl-tRNA or the antibiotic kirromycin, two ligands which were shown previously by cross-linking experiments to be in contact with amino acid residues located in domains 1 and 2, and domain 3, respectively. The results suggest that the isolated domain pair 2 and 3 by itself forms too few contacts with these ligands to form a stable complex. Furthermore, the data suggest that domain 1 in intact EF-Tu, in a subtle but nevertheless decisive manner, alters the conformation of the other two domains in such a way that all three domains cooperatively create a high affinity binding site for aminoacyl-tRNA and the antibiotic kirromycin.
Pieper
U
U
Zentrum Biochemie, Medizinische Hochschule Hannover, F.R.G.
Ehbrecht
H J
HJ
Fliess
A
A
Schick
B
B
Jurnak
F
F
Pingoud
A
A
D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011728:Pyridones / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D006146:Guanidines; D011728:Pyridones; D012346:RNA, Transfer, Amino Acyl; D010444:Peptide Elongation Factor Tu; D019791:Guanidine; C008536:mocimycin