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Comparative genomic and phylogenetic analyses show that homologues of PAB0719 and PAB0760 occur only in a few Archaea, these genes having been acquired via a single horizontal gene transfer from a bacterial donor to the common ancestor of Thermococcales and Nanoarchaea. This transfer event was followed by a duplication event in Thermococcales leading to two closely related genes. None of the gene products of the two P. abyssi paralogues catalyses in vitro the formation of m 5 U in a P. abyssi rRNA fragment homologous to the bacterial RumA substrate. Instead, PAB0719 enzyme (renamed PabTrmU54) displays an identical specificity to TrmA, as it catalyses the in vitro formation of m 5 U-54 in tRNA. Thus, during evolution, at least one of the two P. abyssi RumA-type enzymes has changed of target specificity. This functional shift probably occurred in an ancestor of all Thermococcales. This study also provides new evidence in favour of a close relationship between Thermococcales and Nanoarchaea.

During HIV reverse transcription, (؉) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3 end of tRNA Lys,3 hybridized to the viral primer binding site (PBS). Before (؉) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3 end of primer tRNA Lys,3 . Since the detailed mechanism of (؉) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV Lys,3 . Thus, modified nucleoside m 1 A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (؉) strand DNA transfer. We show that other factors such as the nature of the (؊) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (؉) strand DNA transfer.The distinctive characteristic of retroviruses is the conversion of their RNA genome into double-stranded DNA, which is subsequently integrated into the cellular genome. Reverse transcription is a complex process composed of multiple steps catalyzed by the viral enzyme reverse transcriptase (RT) 1 (reviewed in Ref. 1). The general model of reverse transcription includes two obligatory DNA transfer reactions (Fig. 1A). RT initiates minus strand DNA synthesis by elongation of a specific cellular tRNA primer annealed to the primer binding site (PBS) and continues as far as the 5Ј end of the RNA genome, generating the so-called strong stop cDNA (ss-cDNA). The concomitant degradation of the RNA template by the RNase H activity of RT makes the ss-cDNA single stranded. ss-cDNA is transferred by NC protein (2, 3) to the 3Ј end of either the template RNA or the other RNA molecule present in the virus particle to complete (Ϫ) strand DNA synthesis. (ϩ) strand synthesis is primed by an RNase H-resistant sequence, the polypurine tract (PPT), and continues as far as a doublestranded RNA region corresponding to the 18-nt tRNA-viral PBS RNA complex. RT needs to unwind this tRNA-PBS RNA to reverse-transcribe the 3Ј end of primer tRNA. The stop signal for this (ϩ) strand synthesis has been proposed to be the methylated adenosine at position 58 (m 1 A-58) of primer tRNA (4, 5) but it is not the absolute termination site of RT in the case of HIV (6, 7). Termination at m 1 A-58 results in the (ϩ) strong stop DNA species carrying a complete (ϩ) PBS sequence at its 3Ј terminus. Before (ϩ) strand DNA transfer, the tRNA primer is thought to be removed by the RT-associated RNase H activity (7-11); thus, the (ϩ) PBS at the 3Ј end of (ϩ) ssDNA becomes available for hybridization to the (Ϫ) PBS synthesized at the 3Ј end of the (Ϫ) strand DNA, and synthesis of both strands resumes with strands copying each other (Fig. 1A).In this report, we analyzed the following factors that influence the efficiency of HIV-1 (ϩ) strand DNA transfer: the modified nucleosides of primer tRNA Lys,3 , the RNase H activity...

Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.

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The tRNA:m22G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)—containing N-terminal domain [1–152] and C-terminal catalytic domain [157–329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPα) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPα and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPα structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNAAsp substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.