The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression

Abstract

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio- N 6-isopentenyl-adenosine in position 37, 3′ adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNA Pro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNA Trp, and three species of E. coli tRNA Trp: wild type tRNA Trp (with ms 2i 6A37 † † Abbreviations used: ms 2i 6A, 2-methyl-thio- N 6-isopentenyl-adenosine; V, uridine-5-oxyacetic acid; SDS, sodium dodecyl sulphate and G24), UGA suppressor tRNA Trp (with ms 2i 6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms 2i 6A-deficient as a result of the mutation miaA. Complex formation between tRNA Pro and ms 2i 6A-containing tRNA Trp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms 2i 6A-deficient tRNA Trp makes less stable complexes with tRNA Pro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNA IV Ser (anticodon VGA) replaces tRNA Pro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNA Trp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNA Pro, but are again similar. No complex formation is detectable with the ms 2i 6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Qβ coat protein in vitro. The data on complexes with tRNA Pro suggest that deficiency in ms 2i 6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms 2i 6 stabilizes the anticodon interaction by improved stacking of base 37.