No abstract

Two truncated forms of the Sulfolohus solfuturicus elongation factor 1 a (SsEF-la), corresponding to the putative domains G+M, Ss(GM)EF-la, and G, Ss(G)EF-la, have been constructed by gene engineering, produced in Escherichin coli and purified. Neither truncated form was able to sustain poly(Phe) synthesis but they were able to bind guanine nucleotides with an affinity much higher with respect to that of the intact factor. However, the difference in the affinity for GDP and GTP became progressively reduced with the extent of the truncation. The values of k, , , and K,,, for GTP of the intrinsic GTPase of SsEF-lu triggered by 3.6 M NaCl were not affected by the deletions. In contrast, both Ss(GM)EF-la and Ss(G)EF-la were less thermostable than the intact factor; the region of the factor most responsible for the loss of resistance against heat inactivation was the C-terminal domain. On the other hand the domain M was the regulator of the thermophilicity of SsEF-la since only Ss(G)EF-la showed a reduced thermophilicity. Remarkably, both Ss(GM)EF-la and Ss(G)EF-la were able to exchange ['HIGDP for GTP at a very high rate so that they were no more sensitive to the stimulatory effect of SsEF-Ij?, which is the nucleotide exchange factor of SsEF-I a.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.

No abstract

No abstract