A chimeric elongation factor containing the putative guanine nucleotide binding domain of archaeal EF-1 alpha and the M and C domains of eubacterial EF-Tu

A recombinant chimeric elongation factor containing the region of EF-1R 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 was not required for poly(Phe) synthesis since the chimera was already able to exchange [3H]GDP for GTP at very high rate even at 0 °C. Compared to that of SsEF-1R, the affinity of the chimera for guanine nucleotides was increased and the kcat of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 °C lower than that displayed by SsG and SsEF-1R, respectively, but 30 °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-1R up to 70 °C; at higher temperatures, inactivation occurred.