Mechanism of codon recognition by transfer RNA studied with oligonucleotides larger than triplets.

The binding of yeast tRNAPhe to UUCA, UUCC, UUCCC, UUCUUCU, U4, U5, U6 and U7 was analysed by fluorescence temperature jump and equilibrium sedimentation measurements. In all cases the two observed relaxation processes can be assigned to alpha) an intramolecular conformation change of the anticodon loop and beta) preferential binding of the oligonucleotides to one of the anticodon conformations. The anticodon loop transition is associated with inner sphere complexation of Mg2+ and proceeds with rate constants of about 10(3) s-1. The rate constants of oligonucleotide binding are between 4 and 10 X 10(6) M-1s-1 and reflect an increase of the association rate with the number of binding sites compensated to some degree by electrostatic repulsion in the preequilibrium complex. Neither temperature jump nor equilibrium sedimentation experiments provided evidence for UUCA or UUCC induced tRNA dimerisation, although UUC binding leads to strong tRNA dimerisation under equivalent conditions. The results obtained for the longer oligonucleotides are similar. In the case of UUCUUCU with its two potential binding sites for tRNAPhe there was no evidence for the formation of 'ternary' complexes. Apparently tRNAPhe binds preferentially to the second UUC of this 'messenger' and forms additional contacts with residues on either side of the codon. Some evidence for the formation of ternary complexes is obtained for U6 and U7, although the extent of this reaction remains very small. Our results demonstrate that the mode of tRNA binding to a codon is strongly influenced by residues next to the codon. The formation of cooperative contacts between tRNA molecules at adjacent codons apparently requires support by a catalyst adjusting an appropriate conformation of messenger and tRNA molecules.     

Mechanism of codon recognition by transfer RNA and codon-induced tRNA association

The steps of UUC recognition by tRNAPhe were analysed by temperature-jump measurements. At ion concentrations close to physiological conditions we found three relaxation processes, which we assigned to (1) formation of codon-anticodon complexes, (2) a conformational change of the anticodon loop coupled with Mg2+ binding, and (3) codon-induced association of tRNA. The relaxation data were evaluated both by the usual procedure (fitting the exponentials evaluated from the individual experiments of a set to a reaction model) and by "global fitting", i.e. fitting a set of relaxation curves obtained at various concentrations directly to a reaction model, thus leaving out the intermediate exponential fitting step. The data can be represented quantitatively by a three-step model: the codon binds to the anticodon at a rate of 4 X 10(6) to 6 X 10(6) M-1S-1 as is usual for the formation of oligomer helices; the conformation change of the anticodon loop is associated with inner sphere complexation of Mg2+ at a rate of 10(3) S-1; the codon-tRNA complexes form dimers at a rate of 5 X 10(6) to 15 X 10(6) M-1S-1. A similar mechanism is found for the binding of the wobble codon UUU to tRNAPhe at increased concentrations of Mg2+. Measurements at different Mg2+ concentrations demonstrate the distinct role of this ion in the codon recognition and the codon-induced tRNA dimerization. We propose a simple mechanism, based upon the special properties of magnesium ions, for long-distance transfer of reaction signals along nucleic acid chains.     

Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA^opt) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.     

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA.

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA^Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA^Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA₁^Leu and sequenced tRNA₃^Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA₃^Leu is highly preferred for the CUU codon; tRNA₄^Leu and tRNA₅^Leu both recognize UUA and UUG codons, with tRNA₄^Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX₄ (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX₄ leucine codon series is not paralleled in the UUX₄ leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.