Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop.

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.

Synthesis and investigation of the 5-formylcytidine modified, anticodon stem and loop of the human mitochondrial tRNAMet.

Human mitochondrial methionine transfer RNA (hmtRNA(Met)(CAU)) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f(5)C(34)). The role of this modification in (hmtRNA(Met)(CAU)) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

Preparation and evaluation of acylated tRNAs.

In the cell, the activity of tRNA is governed by its acylation state. Interactions with the ribosome, translation factors, and regulatory elements are strongly influenced by the acyl group, and presumably other cellular components that interact with tRNA also use the acyl group as a specificity determinant. Thus, those using biochemical approaches to study any aspect of tRNA biology should be familiar with effective methods to prepare and evaluate acylated tRNA reagents. Here, methods to prepare aminoacyl-tRNA, N-acetyl-aminoacyl-tRNA, and fMet-tRNA(fMet) and to assess their homogeneity are described. Using these methods, acylated tRNAs of high homogeneity can be reliably obtained.

A novel immobilization method of an active protein via in vitro N-terminal specific incorporation system of nonnatural amino acids.

Recently, we succeeded in incorporating a biotin tag into an active protein, but only at the N-terminal site, in the presence of an Escherichia coli initiator tRNA(fMet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated protein was immobilized on a streptavidin-matrix. We have tried to increase the biotin labeling efficiency (immobilization efficiency) by decreasing concentrations of non-biotinylated tRNA(fMet)s in the translation system.

Requirement of modified residue m1A9 for EF-Tu binding to nematode mitochondrial tRNA lacking the T arm.

Most of nematode mitochondrial (mt) tRNAs lacking the T arm have 1-methyladenosine (m1A) at position 9. To investigate the effect of m1A, we constructed a nematode Ascaris suum mt tRNA(Met) containing only m1A9 as the modified nucleoside by means of molecular surgery. Although the unmodified A. suum mt Met-tRNA(Met) did not bind to nematode mt EF-Tu, the m1A9-containing tRNA bound to the EF-Tu, suggesting that m1A at position 9 is necessary for binding of nematode mt tRNAs lacking the T arm to the EF-Tu, probably because of maintenance of the L-shape-like structure or interaction with the C-terminal amino acid residues of the EF-Tu.

Preparation of biologically active Ascaris suum mitochondrial tRNAMet with a TV-replacement loop by ligation of chemically synthesized RNA fragments.

Ascaris suum mitochondrial tRNA Met lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification. Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA Met, which has already been concluded to take an L-shape-like structure [Watanabe et al. (1994) J. Biol. Chem., 269, 22902-22906]. These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.

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.

Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNA(Met).

A 75-unit long oligoribonucleotide corresponding to the sequence of the Saccharomyces cerevisiae initiator tRNA was synthesized chemically. The crude RNA was purified, and the sequence was verified by RNA sequencing techniques. A particularly useful purification step involved hydrophobic chromatography on BND-cellulose. The purified RNA could be aminoacylated to 28% of a bona fide initiator tRNA(Met) sample and threonylated to 76% of the level observed with native tRNA(fMet) from E. coli.

Solid phase synthesis of the 5'-half of the initiator t-RNA from B. subtilis.

Using cyanoethyldiisopropylamino phosphoramidite chemistry, four oligonucleotides constituting a part of the sequence of the initiator t-RNA from B. subtilis were synthesized. For the protection of the exocyclic amino functions of bases, phenoxyacetyl group was used for adenine and guanine, and acetyl group was preferred for cytosine. With these labile groups, final deprotection of the oligonucleotides can be performed in milder conditions, allowing the incorporation of 5,6-dihydrouridine in a 35-mer constituting the 5'-end of the t-RNA.

Labile protecting groups in tRNA synthesis.

The use of labile protecting groups for the protection of the exocyclic amino function of adenine, guanine and cytosine has two main advantages in RNA synthesis. Final deprotection in concentrated aqueous ammonia takes place in milder conditions which are more compatible with the sensitivity of oligoribonucleotides towards alkali-conditions. The introduction of fragile bases such as certain modified bases encountered in the primary structure of t-RNA is feasible. The chemical synthesis of RNA fragments constituting the primary structure of B. subtilis f-methionine t-RNA is described.

Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.

Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47.