tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence.

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria-which models tuberculous granulomas-are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA(Leu(UUR)) and tRNA(Lys).

5-Taurinomethyluridine (τm(5)U) and 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) are located at the wobble position of human mitochondrial (hmt) tRNA(Leu(UUR)) and tRNA(Lys), respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys) are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm(5)U and τm(5)s(2)U into 17-mers related to the sequence of the anticodon arms hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys), respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF3) are compatible with the blockage of the canonical monomeric units. The synthesis of τm(5)s(2)U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.

Site-selected incorporation of 5-carboxymethylaminomethyl(-2-thio)uridine into RNA sequences by phosphoramidite chemistry.

5-Carboxymethylaminomethyluridine (cmnm(5)U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U) are located at the wobble position in several cytosolic and mitochondrial tRNA sequences. In this paper, we report the first site-selected incorporation of cmnm(5)U and cmnm(5)s(2)U into RNA sequences by phosphoramidite chemistry on a CPG solid support. Trifluoroacetyl and 2-(trimethylsilyl)ethyl were selected for the protection of the amine and carboxyl functions, respectively.

mt-tRNA components: synthesis of (2-thio)uridines modified with blocked glycine/taurine moieties at C-5,1.

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2',3'-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)3) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm(5)(s(2))U) and 5-taurinomethyl(-2-thio)uridine (τm(5)(s(2))U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2',3'-O-isopropylidene-2-thiouridine is also reported.

Human tRNA(Lys3)(UUU) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism.

Human tRNA(Lys3)(UUU) (htRNA(Lys3)(UUU)) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)), 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A(37)), and pseudouridine (Ψ(39)) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39)). Ribosome binding assays in vitro revealed that the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound AAA and AAG codons, whereas binding of the unmodified ASL(Lys3)(UUU) was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ(39) enhanced the thermodynamic stability of the ASL through base stacking while ms(2)t(6)A(37) restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm(5)s(2)U(34) and ms(2)t(6)A(37) participate in the stability of the anticodon-codon interaction. Importantly, the mcm(5)s(2)U(34)·G(3) wobble base pair is in the Watson-Crick geometry, requiring unusual hydrogen bonding to G in which mcm(5)s(2)U(34) must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons.

Modifications modulate anticodon loop dynamics and codon recognition of E. coli tRNA(Arg1,2).

Three of six arginine codons are read by two tRNA(Arg) isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASL(Arg1,2)) differs only in that the position 32 cytidine of tRNA(Arg1) is posttranscriptionally modified to 2-thiocytidine (s(2)C(32)). The tRNA(Arg1,2) are also modified at positions 34 (inosine, I(34)) and 37 (2-methyladenosine, m(2)A(37)). To investigate the roles of modifications in the structure and function, we analyzed six ASL(Arg1,2) constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASL(Arg1,2) showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASL(Arg1,2) constructs efficiently bound the cognate CGU codon. Three ASLs with I(34) were able to decode CGC, whereas only the singly modified ASL(Arg1,2)(ICG) with I(34) was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4-1.4 µM). However, with the introduction of s(2)C(32) or m(2)A(37) to ASL(Arg1,2)(ICG), the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASL(Arg1,2), which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons.

3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine is one of two novel post-transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei.

tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications, many of which are chemically similar. Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys)(UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher-order tandem MS. Phylogenetic comparison with modifications in tRNA(Lys) from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNA(Lys)(UUU), where U47 exhibited a novel modification, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2-methylthio-N(6)-threonylcarbamoyl-modification, and a major fraction with A37 being modified by a 294.0-Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Functional recognition of the modified human tRNALys3(UUU) anticodon domain by HIV's nucleocapsid protein and a peptide mimic.

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA(Lys3)(UUU) as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA(Lys3)(UUU), the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL(Lys3)) were studied. NCp7 bound the hASL(Lys3)(UUU) modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm(5)s(2)U(34)) and 2-methylthio-N(6)-threonylcarbamoyladenosine at position-37 (ms(2)t(6)A(37)) with a considerably higher affinity than the unmodified hASL(Lys3)(UUU) (K(d)=0.28±0.03 and 2.30±0.62 µM, respectively). NCp7 denatured the structure of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) more effectively than that of the unmodified hASL(Lys3)(UUU). Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K(d)=0.55±0.10 µM) and specificity for the ASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37) comparable to that of NCp7. The peptides recognized a t(6)A(37)-modified ASL with an affinity (K(d)=0.60±0.09 µM) comparable to that for hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37), indicating a preference for the t(6)A(37) modification. Significantly, one of the peptides was capable of relaxing the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA(Lys3)(UUU) have been found to be important determinants of NCp7's recognition prior to the tRNA(Lys3)(UUU) being annealed to the viral genome as the primer of reverse transcription.

ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.

Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

The structure of the human tRNALys3 anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs.

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

Anticodon domain modifications contribute order to tRNA for ribosome-mediated codon binding.

The accuracy and efficiency with which tRNA decodes genomic information into proteins require posttranscriptional modifications in or adjacent to the anticodon. The modification uridine-5-oxyacetic acid (cmo (5)U 34) is found at wobble position 34 in a single isoaccepting tRNA species for six amino acids, alanine, leucine, proline, serine, threonine, and valine, each having 4-fold degenerate codons. cmo (5)U 34 makes possible the decoding of 24 codons by just six tRNAs. The contributions of this important modification to the structures and codon binding affinities of the unmodified and fully modified anticodon stem and loop domains of tRNA (Val3) UAC (ASL (Val3) UAC) were elucidated. The stems of the unmodified ASL (Val3) UAC and that with cmo (5)U 34 and N (6)-methyladenosine, m (6)A 37, adopted an A-form RNA conformation (rmsd approximately 0.6 A) as determined with NMR spectroscopy and torsion-angle molecular dynamics. However, the UV hyperchromicity, circular dichroism ellipticity, and structural analyses indicated that the anticodon modifications enhanced order in the loop. ASL (Val3) UAC-cmo (5)U 34;m (6)A 37 exhibited high affinities for its cognate and wobble codons GUA and GUG, and for GUU in the A-site of the programmed 30S ribosomal subunit, whereas the unmodified ASL (Val3) UAC bound less strongly to GUA and not at all to GUG and GUU. Together with recent crystal structures of ASL (Val3) UAC-cmo (5)U 34;m (6)A 37 bound to all four of the valine codons in the A-site of the ribosome's 30S subunit, these results clearly demonstrate that the xo (5)U 34-type modifications order the anticodon loop prior to A-site codon binding for an expanded codon reading, possibly reducing an entropic energy barrier to codon binding.

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines.

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

Specificity of phage display selected peptides for modified anticodon stem and loop domains of tRNA.

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.

The role of modifications in codon discrimination by tRNA(Lys)UUU.

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation.

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.

Accurate translation of the genetic code depends on tRNA modified nucleosides.

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.