Activities of guanosine triphosphate analogues in reactions catalyzed by elongation factor Tu and initiation factor 2 of Escherichia coli

Abstract

In earlier studies two natural analogues of GTP, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and dGTP, were found to substitute for GTP in reactions catalyzed by initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu), while only dGTP could replace GTP with elongation factor G. These observations with IF-2 and EF-Tu have been extended to two analogues of GTP modified at the 3′ ribose hydroxyl position, 3′-deoxyguanosine 5′-triphosphate (3′dGTP) and 3′-deoxy-3′-aminoguanosine 5′-triphosphate (3′dNH 2GTP). These compounds were found to be similar to GTP, dGTP, and pppGpp in IF-2-dependent formation of N- formylmethionyl-puromycin and EF-Tu-dependent formation of N- acetyl-Phe-Phe-tRNA . The apparent K m values for the five guanosine nucleotides were 2 · 10 −6–4 · 10 −6 M in the former reaction and 2 · 10 −7–6 · 10 −7 M in the latter. These reactions did not have an absolute requirement for either an intact pentose ring or for the guanine base in the nucleotide. Although substantially less active than the guanine nucleotides, ITP and the dialcohol derived from GTP by periodate oxidation and borohydride reduction (ox-redGTP) were partially active in both the IF-2- and EF-Tu-dependent reactions, with apparent K m values about 40–100 times those of GTP. IDP and periodate-oxidized, borohydride-reduced GTP were found to inhibit both the IF-2- and EF-Tu-dependent reactions, but much less effectively than GDP. When a 5′-diphosphate was inhibiting the homologous 5′-triphosphate, however, the results with all compounds were similar. The binding of nucleotides to EF-Tu was also examined. The guanine nucleotides were comparable, but a much weaker binding could also be demonstrated with ITP and oxredGTP.