Umeå universitets logga

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo⁵U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons.

To study the function of cmo⁵U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo⁵U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo⁵U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo⁵U is different for the different tRNAs. In addition, we found that cmo⁵U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons.

The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNA^Pro_[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm⁵s²U34. The presence of an extra sidechain (C₁₀H₁₇) on the wobble nucleoside of tRNA^Gln_[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNA^Pro_[cmo5UGG] to slip into the +1 frame.

We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNA^Pro_[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.

Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm⁵s²U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo⁵U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described.

A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C.

cmo⁵U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo⁵U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo⁵U containing tRNA present in the cell. The influence of cmo⁵U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent.

During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNA^Gln_cmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm⁵s²U34. The presence of UK on tRNA^Gln_U*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.