Anticodon loop mutations perturb reading frame maintenance by the E site tRNA

The ribosomal E site helps hold the reading frame. Certain tRNA mutations affect translation, and anticodon loop mutations can be especially detrimental. We studied the effects of mutations saturating the anticodon loop of the amber suppressor tRNA, Su7, on the ability to help hold the reading frame when in the E site. We also tested three mutations in the anticodon stem, as well as a mutation in the D stem (the “Hirsh” mutation). We used the Escherichia coli RF2 programmed frameshift site to monitor frame maintenance. Most anticodon loop mutations increase frameshifting, possibly by decreasing codon:anticodon stability. However, it is likely that the A site is more sensitive to anticodon loop structure than is the E site. Unexpectedly, the Hirsh mutation also increases frameshifting from the E site. Other work shows that mutation may increase the ability of tRNA to react in the A site, possibly by facilitating conformational changes required for aminoacyl-tRNA selection. We suggest that this property may decrease its ability to bind to the E site. Finally, the absence of the ms²io⁶A nucleoside modifications at A37 does not decrease the ability of tRNA to help hold the reading frame from the E site. This was also unexpected because the absence of these modifications affects translational properties of tRNA in A and P sites. The absence of a negative effect in the E site further highlights the differences among the substrate requirements of the ribosomal coding sites.     

Conformation effects of base modification on the anticodon stem-loop of Bacillus subtilis tRNA(Tyr)

tRNA molecules contain 93 chemically unique nucleotide base modifications that expand the chemical and biophysical diversity of RNA and contribute to the overall fitness of the cell. Nucleotide modifications of tRNA confer fidelity and efficiency to translation and are important in tRNA-dependent RNA-mediated regulatory processes. The three-dimensional structure of the anticodon is crucial to tRNA-mRNA specificity, and the diverse modifications of nucleotide bases in the anticodon region modulate this specificity. We have determined the solution structures and thermodynamic properties of Bacillus subtilis tRNA^Tyr anticodon arms containing the natural base modifications N⁶-dimethylallyl adenine (i⁶A₃₇) and pseudouridine (ψ₃₉). UV melting and differential scanning calorimetry indicate that the modifications stabilize the stem and may enhance base stacking in the loop. The i⁶A₃₇ modification disrupts the hydrogen bond network of the unmodified anticodon loop including a C₃₂-A₃₈⁺ base pair and an A₃₇-U₃₃ base-base interaction. Although the i⁶A₃₇ modification increases the dynamic nature of the loop nucleotides, metal ion coordination reestablishes conformational homogeneity. Interestingly, the i⁶A₃₇ modification and Mg²⁺ are sufficient to promote the U-turn fold of the anticodon loop of Escherichia coli tRNA^Phe, but these elements do not result in this signature feature of the anticodon loop in tRNA^Tyr.     

Exploring GpG bases next to anticodon in tRNA subsets

Transfer RNA (tRNA) structure, modifications and functions are evolutionary and established in bacteria, archaea and eukaryotes. Typically the tRNA modifications are indispensable for its stability and are required for decoding the mRNA into amino acids for protein synthesis. A conserved methylation has been located on the anticodon loop specifically at the 37^th position and it is next to the anticodon bases. This modification is called as m1G37 and it is catalyzed by tRNA (m¹G37) methyltransferase (TrmD). It is deciphered that G37 positions occur on few additional amino acids specific tRNA subsets in bacteria. Furthermore, Archaea and Eukaryotes have more number of tRNA subsets which contains G37 position next to the anticodon and the G residue are located at different positions such as G36, G37, G38, 39, and G40. In eight bacterial species, G (guanosine) residues are presents at the 37^th and 38^th position except three tRNA subsets having G residues at 36^th and 39^th positions. Therefore we propose that m1G37 modification may be feasible at 36^th, 37^th, 38^th, 39^th and 40^th positions next to the anticodon of tRNAs. Collectively, methylation at G residues close to the anticodon may be possible at different positions and without restriction of anticodon 3^rd base A, C, U or G.     

Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity

tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.     

Structural insights into translational recoding by frameshift suppressor tRNASufJ

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA^SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA^SufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL^SufJ or tRNA^SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL^SufJ and ASL^Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL^SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA^SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.     

Determination of N-(9-( -D-ribofuranosyl)purin-6-ylcarbamoly)threonine at the picomole level in transfer-RNA

An assay has been developed for quantitation of the modified nucleoside, t⁶A, in tRNA at the pmole level. For tRNA from a variety of species, the content of t⁶A was found to be 0.18–0.25 mole %. These values lend support to the suggestion that t⁶A is located at the 3′-end of the anticodon in tRNA's whose codons begin with adenosine. Essentially no t⁶A was found in Mycoplasma sp. (Kid) tRNA which is deficient in many modified nucleosides. In the rat, no organ specific differences were found. The amount of t⁶A in Novikoff hepatoma tRNA was essentially the same as in tRNA from normal rat liver.     

Functional Analysis of Human tRNA Isodecoders

tRNA isodecoders share the same anticodon but have differences in their body sequence. An unexpected result from genome sequencing projects is the identification of a large number of tRNA isodecoder genes in mammalian genomes. In the reference human genome, more than 270 isodecoder genes are present among the approximately 450 tRNA genes distributed among 49 isoacceptor families. Whether sequence diversity among isodecoder tRNA genes reflects functional variability is an open question. To address this, we developed a method to quantify the efficiency of tRNA isodecoders in stop-codon suppression in human cell lines. First, a green fluorescent protein (GFP) gene that contains a single UAG stop codon at two distinct locations is introduced. GFP is only produced when a tRNA suppressor containing CUA anticodon is co-transfected with the GFP gene. The suppression efficiency is examined for 31 tRNA isodecoders (all contain CUA anticodon), 21 derived from four isoacceptor families of tRNA^Ser genes, 7 from five families of tRNA^Leu genes, and 3 from three families of tRNA^Ala genes. We found that isodecoder tRNAs display a large difference in their suppression efficiency. Among those with above background suppression activity, differences of up to 20-fold were observed. We were able to tune tRNA suppression efficiency by subtly adjusting the tRNA sequence and inter-convert poor suppressors into potent ones. We also demonstrate that isodecoder tRNAs with varying suppression efficiencies have similar stability and exhibit similar levels of aminoacylation in vivo. Our results indicate that naturally occurring tRNA isodecoders can have large functional variations and suggest that some tRNA isodecoders may perform a function distinct from translation.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA^Opt. We suspected a modification of the tRNA^Opt_AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA^Opt_AUG is converted to inosine. We identified tRNA^Opt_AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.     

Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons

The recessive lethal amber suppressor su⁺7(UAG-1) in Escherichia coli inserts glutamine in response to the UAG codon. The genetic analysis presented in this paper shows that the su^?7 precursor allele can give rise to suppressors of the UGA codon as well as of the UAG codon. This observation suggests that the su^?7 gene normally codes for transfer RNA^Trp, a tRNA whose anticodon can be modified by single base changes to forms that can translate either UAG or UGA. The chemical findings presented in the accompanying paper (Yaniv et al., 1974) are wholly in accord with this interpretation. Thus, a single base substitution in the anticodon sequence of a tRNA can affect both the coding specificity of the molecule and also the amino acid acceptor specificity.     

The naturally occurring N6-threonyl adenine in anticodon loop of Schizosaccharomyces pombe tRNAi causes formation of a unique U-turn motif

Modified nucleosides play an important role in structure and function of tRNA. We have determined the solution structure of the anticodon stem–loop (ASL) of initiator tRNA of Schizosaccharomyces pombe. The incorporation of N6-threonylcarbamoyladenosine at the position 3′ to the anticodon triplet (t⁶A37) results in the formation of a U-turn motif and enhances stacking interactions within the loop and stem regions (i.e. between A35 and t⁶A37) by bulging out U36. This conformation was not observed in a crystal structure of tRNAi including the same modification in its anticodon loop, nor in the solution structure of the unmodified ASL. A t⁶A modification also occurs in the well studied anti-stem–loop of lys-tRNA_UUU. A comparison of this stem–loop with our structure demonstrates different effects of the modification depending on the loop sequence.     

tRNA anticodon shifts in eukaryotic genomes

Embedded in the sequence of each transfer RNA are elements that promote specific interactions with its cognate aminoacyl tRNA-synthetase. Although many such “identity elements” are known, their detection is difficult since they rely on unique structural signatures and the combinatorial action of multiple elements spread throughout the tRNA molecule. Since the anticodon is often a major identity determinant itself, it is possible to switch between certain tRNA functional types by means of anticodon substitutions. This has been shown to have occurred during the evolution of some genomes; however, the scale and relevance of “anticodon shifts” to the evolution of the tRNA multigene family is unclear. Using a synteny-conservation–based method, we detected tRNA anticodon shifts in groups of closely related species: five primates, 12 Drosophila, six nematodes, 11 Saccharomycetes, and 61 Enterobacteriaceae. We found a total of 75 anticodon shifts: 31 involving switches of identity (alloacceptor shifts) and 44 between isoacceptors that code for the same amino acid (isoacceptor shifts). The relative numbers of shifts in each taxa suggest that tRNA gene redundancy is likely the driving factor, with greater constraint on changes of identity. Sites that frequently covary with alloacceptor shifts are located at the extreme ends of the molecule, in common with most known identity determinants. Isoacceptor shifts are associated with changes in the midsections of the tRNA sequence. However, the mutation patterns of anticodon shifts involving the same identities are often dissimilar, suggesting that alternate sets of mutation may achieve the same functional compensation.     

Discovery and characterization of tRNA lysidine synthetase (TilS)

In the bacterial decoding system, the AUA codon is deciphered as isoleucine by tRNA^Ile bearing lysidine (L, 2-lysyl-cytidine) at the wobble position. Lysidine is an essential modification that determines both the codon and amino acid specificities of tRNA^Ile. We identified an enzyme named tRNA^Ile lysidine synthetase (TilS) that catalyzes lysidine formation by using lysine and ATP as substrates. Biochemical studies revealed a molecular mechanism of lysidine formation that consists of two consecutive reactions involving the adenylated tRNA intermediate. In addition, we deciphered how Escherichia coli TilS specifically discriminates between tRNA^Ile and the structurally similar tRNA^Met, which bears the same anticodon loop. Recent structural studies unveiled tRNA recognition by TilS, and a molecular basis of lysidine formation at atomic resolution.