Site-directed mutagenesis on the translation elongation factors from the archaeon Sulfolobus solfataricus

The present article reviews our recent work carried out on the translation elongation factors EF-1alpha, EF-2 and EF-1beta isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. During the elongation cycle the GTPase activity of SsEF-1alpha and SsEF-2 and the nucleotide exchange activity of SsEF-1beta are crucial reactions ensuring the efficiency and the unidirectionality of the entire protein synthesis process. The site-directed mutagenesis carried out on the first consensus motif [G/A]XXXXGK of SsEF-1alpha and SsEF-2 allowed to assess the key role played by the alternate presence of G or A in the first position of this sequence motif. In particular, the data reported indicate that the presence of glycine triggers the intrinsic GTPase of both elongation factors, whereas alanine keeps this activity to a minimum level. The mutagenic analysis of a conserved valine faced to the above mentioned glycine in SsEF-1alpha showed its involvement in the interaction with aa-tRNA and in the stability of the enzyme. Even in S. solfataricus the nucleotide exchange activity of SsEF-1beta is essential to recycle the active GTP-bound form of SsEF-1alpha. However, some relevant structural and functional differences emerge, when comparing the properties of SsEF-1beta with those of eubacterial and eukaryal functional analogues. The smaller size of SsEF-1beta and its transient interaction with the SsEF-1alpha-GDP complex represent distinctive properties of the archaeal exchange factor. In conclusion, the data reported indicate that the general scheme of the elongation cycle is conserved also in the hyperthermophilic source analysed, even though the structural properties and the intermediate reactions catalysed by the elongation factors may be slightly different in different species.