Efficient conversion of N6-threonylcarbamoyladenosine (t6A) into a tRNA native hydantoin cyclic form (ct6A) performed at nucleoside and oligoribonucleotide levels.

A t6A nucleoside was efficiently and stereospecifically transformed into a hydantoin cyclic form of N6-l-threonylcarbamoyladenosine (ct6A) by the use of polymer bounded carbodiimide (EDC-P) and HOBt. The procedure was successfully applied for a post-synthetic conversion of t6A-containing RNA 17-mers (of the sequences of anticodon stem and loop (ASL) fragments of S. pombe tRNAi and E. coli tRNALys) into the products bearing the ct6A unit.

Synthesis of the tRNA(Lys,3) anticodon stem-loop domain containing the hypermodified ms2t6A nucleoside.

The synthesis of a protected form of the hypermodified nucleoside, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNA(Lys,3), the primer for HIV-1 reverse transcriptase. Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t6A containing E. coli tRNA(Lys), despite the presence of the bulky methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA-RNA interactions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.

Synthesis and biophysical characterization of tRNA(Lys,3) anticodon stem-loop RNAs containing the mcm(5)s(2)U nucleoside.

[reaction: see text] Phosphoramidite reagents of the naturally occurring modified nucleosides mcm(5)s(2)U and mcm(5)U were synthesized and along with pseudouridine were incorporated into 17-nucleotide lysine tRNA anticodon stem-loop domains. Standard RNA phosphoramidite coupling chemistry allowed us to systematically investigate the thermodynamic effects of nucleoside modification and to correlate thermodynamic trends with qualitative structure effects seen by NMR spectroscopy.

Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A.

The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.

Synthesis and characterization of the native anticodon domain of E. coli TRNA(Lys): simultaneous incorporation of modified nucleosides mnm(5)s(2)U, t(6)A, and pseudouridine using phosphoramidite chemistry.

The anticodon domain of E. coli tRNA(Lys) contains the hypermodified nucleosides mnm(5)s(2)U and t(6)A at positions 34 and 37, respectively, along with a more common psi at position 39. The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature. 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm(5)s(2)U and t(6)A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis. The 17 nucleotide anticodon stem-loop of E. coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1. 8 mg of purified RNA from a 1 micromol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting. The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.

A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys.

We have previously shown by chemical and enzymatic structure probing that, opposite to the native human mitochondrial tRNA(Lys), the corresponding in vitro transcript does not fold into the expected tRNA-specific cloverleaf structure. This RNA folds into a bulged hairpin, including an extended amino acid acceptor stem, an extra large loop instead of the T-stem and loop, and an anticodon-like domain. Hence, one or several of the six modified nucleotides present in the native tRNA are required and responsible for its cloverleaf structure. Phylogenetic comparisons as well as structural analysis of variant transcripts had pointed to m(1)A9 as the most likely important modified nucleotide in the folding process. Here we describe the synthesis of a chimeric tRNA(Lys) with m(1)A9 as the sole modified base and its structural analysis by chemical and enzymatic probing. Comparison of this structure to that of the unmodified RNA, the fully modified native tRNA, and a variant designed to mimic the effect of m(1)A9 demonstrates that the chimeric RNA folds indeed into a cloverleaf structure that resembles that of the native tRNA. Thus, due to Watson-Crick base-pair disruption, a single methyl group is sufficient to induce the cloverleaf folding of this unusual tRNA. This is the first direct evidence of the role of a modified nucleotide in RNA folding.

Structural constraints in the HIV-1 reverse transcriptase-primer/template complex for the initiation of DNA synthesis from primer tRNALys3.

The topography and functional implications of the complex formed in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNALys3 were studied in this work. On the basis of previous results showing the high affinity both of the native primer, tRNALys3, as well as that of mismatched short oligonucleotide primers for HIV-1 RT, we synthesized chimeric primers containing tRNALys3 linked to U and T residues of different lengths. We found that the affinity of the oligonucleotide primers for HIV-1 RT is dramatically increased when linked to primer tRNA. Our results also show that in the tRNA.RT complex, before annealing tRNALys3 to the retroviral RNA genome, the 3'-terminal nucleotide of tRNALys3 is positioned at a distance of one nucleotide unit away from the template in the active polymerization site of the enzyme.