Temperature Jump Relaxation Studies on the Interactions Between Transfer RNAs with Complementary Anticodons. The Effect of Modified Bases Adjacent to the Anticodon Triplet

Abstract

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.

Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner

“…A37 stabilises the intrinsically weak interaction of the A36-U base-pair (Jukes, 1973;Nishimura, 1972). Indeed, the presence of the ms 2 i 6 A37 modi®cation stabilises tRNA-tRNA dimers with complementary anticodons mainly due to an improved stacking of the hypermodi®ed nucleoside (Houssier & Grosjean, 1985;Vacher et al, 1984). Thus, the results from such model experiments suggest that the modi®cation of A37 to ms 2 io 6 A37 stabilises the 3 H stacking features of the anticodon and thereby improves its interaction with the codon.…”

The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA

“…In model experiments in which dissociation of tRNA dimers with complementary anticodons was monitored, it was shown that this modification stabilizes anticodon-anticodon interaction by increased stacking (63). Such experiments have also shown that the ms 2 group is the major stabilizing factor (39). Thus, the effects observed in the aforementioned in vivo and in vitro experiments with tRNA from a miaA mutant were expected to be primarily due to lack of the ms 2 group.…”

The transition from noncoded to coded protein synthesis: did coding mRNAs arise from stability-enhancing binding partners to tRNA?

“…Interactions between E. coli tRNA Gly (with anticodon U*CC, where U* is modified) and tRNA Ser (with an unmodified GGA anticodon) were found to be highly stable [73]; the sole anticodon loop modification in this interaction is that of U34 in the first (wobble) position of the tRNA Gly anticodon, as indicated. Importantly, neither tRNA has a modified purine at position 37, leading the authors to state that, "the presence of three consecutive purines in the [tRNA Ser ] anticodon triplet together with the two purines on its 3' side may yield sufficient stability..." [74]. The interaction between wheat tRNA Gly and E. coli tRNA Ala (with unmodified GCC and GGC anticodons respectively) is also highly stable [75].…”