Synthesis of 3'-fluoro-tRNA analogues for exploring non-ribosomal peptide synthesis in bacteria.

Aminoacyl-tRNAs (aa-tRNAs) participate in a vast repertoire of metabolic pathways, including the synthesis of the peptidoglycan network in the cell walls of bacterial pathogens. Synthesis of aminoacyl-tRNA analogues is critical for further understanding the mechanisms of these reactions. Here we report the semi-synthesis of 3'-fluoro analogues of Ala-tRNA(Ala) . The presence of fluorine in the 3'-position blocks Ala at the 2'-position by preventing spontaneous migration of the residue between positions 2' and 3'. NMR analyses showed that substitution of the 3'-hydroxy group by fluorine in the ribo configuration favours the S-type conformation of the furanose ring of terminal adenosine A76. In contrast, the N-type conformation is favoured by the presence of fluorine in the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. These compounds should provide insight into substrate recognition by Fem transferases and the Ala-tRNA synthetases.

Synthesis and biological evaluation of non-isomerizable analogues of Ala-tRNA(Ala).

Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions. Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNA(Ala) containing an amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine in the natural substrate. The non-isomerizable 2' and 3' regioisomers were not used as substrates by FemX(Wv), an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50 of 5.8 and 5.5 µM, respectively.

Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies.

The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.

[Total chemical synthesis of natural transfer RNA]. / Synthèse chimique totale d'un ARN de transfert naturel.

New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.

Further improvement in protecting method for the oligonucleotide synthesis in terms of a cellulose acetate derivative as a polymer support.

For further improvement in the investigation to utilize a cellulose acetate derivative as a novel type of polymer-support for the synthesis of oligonucleotides, the investigations on utilizing another spacer; on protecting groups for O6-position of guanosine unit, ribothymidine, and pseudouridine; and on a novel protecting group for the introduction of phosphate function at 5'-terminal position, targeting the syntheses of 13-mer, ApApGpGpApApApApUpUpApUpG, 11-mer, pCpUpCpGpUpCpCpApCpCpA, and 12-mer, UpCpCpGpGprTp- psipCpGpApUpU, found in the partial structures of a yeast tRNA(Ala), will be described in detail.

Biological function of modified nucleotides in tRNA molecules--synthesis and biological activity of the analogues of yeast alanyl tRNA with I34 replaced by A34 or G34.

Analogues of yeast alanyl tRNA with I34 replaced by A34 or G34 were synthesized. Synthetic analogues of yeast alanyl tRNT occupy the same position as the natural yeast alanyl tRNA on polyacrylamide gel electrophoresis, and their purity is about 95% after electrophoresis on a 10% or 20% polyacrylamide gel. The two terminal and nearest neighbour nucleotides of the analogues are all correct. The accepting activity of the synthetic analogues is similar to that of the reconstituted natural yeast alanyl tRNA. The incorporation activity of alanine into proteins of the synthetic analogues is about 30% of that of the natural of reconstituted natural yeast alanyl tRNA when I34 is replaced by A, and is 90% when I34 is replaced by G. The reason of the variation in biological function of the analogues of yeast alanyl tRNA after I34 replaced by A or G was discussed.

Chemical synthesis of a biologically active natural tRNA with its minor bases.

The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.