Properties of the elongation factor 1α in the thermoacidophilic archaebacterium Sulfolobus solfataricus

Abstract

The elongation factor 1 a (aEF-la) was purified to homogeneity from the thermoacidophilic archaebacterium Suljolobus soljataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1 : 1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-la is the counterpart of the eubacterial elongation factor Tu (EF-Tu).Purified aEF-la is a monomeric protein with a relative molecular mass of 49000 as determined by SDSjPAGE and by gel filtration on Sephadex G-300; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-la from Artemia salina or of archaebacterial EF-la from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared.aEF-1 a shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95°C; it is also quite heat-resistant, since after a 6-h exposure at 60°C and 87°C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively.The affinity of aEF-la for GDP and GTP was also evaluated. At 80°C Kb for GDP was about 30-fold higher than Ki for GTP; at the same temperature Ki for GDP was 1.7 pM and Ki for GTP was 50 pM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30°C; compared to the values at 0°C of EF-Tu from E. coli and T. thermophilus or EF-la from A . salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors. At 80°C kL GDP and k ; GDP of aEF-la were 7.8 min-' and 4.6 min-' . pM-', respectively; comparison with the values attributed to E. coli EF-Tu at 30°C indicates that the dissociation rate constant increases and the association rate constant decreases, thus indicating that the higher Ki for GDP of aEF-la is due to a synchronous alteration of the rates of both the dissociation and the association processes between GDP and aEF-1 a.Besides aEF-la, the other two elongation factors, aEF-2 and aEF-lP were also present in S. solfataricus, as suggested by the occurrence of reactions typical of each of them. This allows the hypothesis that in this extremophilic archaebacterium the protein synthesis process follows the same general pattern found in all other living organisms.Extremophilic archaebacteria live under unusual conditions of temperature, pH and salt concentration. Their proteins are receiving increasing attention since they are characterized by a great resistance to chemical and physical agents which are deleterious to the homologous proteins isolated from organisms living under normal conditions [l -31....

Studies on the Polypeptide Elongation Factor 2 from Sulfolobus solfataricus

“…In the present report we show that SsEF-2 is able to bind guanosine nucleotides and to promote the hydrolysis of GTP in the presence of ribosomes as well. These findings support the hypothesis that the translocation step in the elongation of the polypeptide chain in archaeal systems involves similar mechanisms as those described for eukarya and eubacteria (1,(5)(6)(7) 2 , 0.05 mg/ml poly(U), 1 mM spermine, 1 mM ATP.…”

Properties of Truncated Forms of the Elongation Factor 1α from the Archaeon Sulfolobus solfataricus

“…GDP and Ss(G)EF-la . GDP complexes were determined as already reported (Masullo et al, 1991); k:, was derived from the equation k: = kY,/K:l.…”

“…The isolation of total tRNA and ribosomes from S. solfataricus and the poly(U)-directed poly(Phe) synthesis were performed as already reported (Masullo et al, 1991). Nucleotide sequencing of the gene constructs was as described .…”

“…The ability of the intact and truncated forms of SsEF-la to form a binary complex with ['HIGDP was assayed as described (Masullo et al, 1991 the number of ['HIGDP binding sites and k! of the Ss(GM)EFl a .…”

“…It belongs to the family of the GTP-binding proteins and binds GDP or GTP at 1 : 1 molar ratio (Masullo et al, 1991); in addition, SsEF-lrx is able to elicit an intrinsic GTPase activity which is triggered by NaCl at molar concentration (GTPase"") (Masullo et al, 1994). The ['HIGDP binding ability and GTPaseN" of SsEF-lcr show a maximum of activity at temperatures above 90°C; for both activities the half-life at 95°C is 10 min (Masullo et al, 1991(Masullo et al, , 1993Ianniciello et al, 1996).…”