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-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.
Arcari
P
P
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
Masullo
M
M
Arcucci
A
A
Ianniciello
G
G
de Paola
B
B
Bocchini
V
V
D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D046911:Macromolecular Substances; D008958:Models, Molecular; D010452:Peptide Biosynthesis / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016944:Sulfolobus / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D014316:Tritium
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D020648:Peptide Elongation Factor 1; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014316:Tritium; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu