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

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-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 tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, 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 tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A 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 ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.