Specific function of a G.U wobble pair from an adjacent helical site in tRNA(Ala) during recognition by alanyl-tRNA synthetase.

G.U wobble pairs are crucial to many examples of RNA-protein recognition. We previously concluded that the G.U wobble pair in the acceptor helix of Escherichia coli alanine tRNA (tRNA(Ala)) is recognized indirectly by alanyl-tRNA synthetase (AlaRS), although direct recognition may play some role. Our conclusion was based on the finding that amber suppressor tRNA Ala with G.U shifted to an adjacent helical site retained substantial but incomplete Ala acceptor function in vivo. Other researchers concluded that only direct recognition is operative. We report here a repeat of our original experiment using tRNA(Lys) instead of tRNA(Ala). We find, as in the original experiment, that a shifted G.U confers Ala acceptor activity. Moreover, the modified tRNA(Lys) was specific for Ala, corroborating our original conclusion and making it more compelling.     

Translocation within the acceptor helix of a major tRNA identity determinant

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl-tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific-a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNA(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.     

Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition

Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs. A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant. Further understanding has been limited due to lack of a crystal structure of the enzyme. We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS. It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold. Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data. It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s.     

Efficient aminoacylation of the tRNA(Ala) acceptor stem: dependence on the 2:71 base pair

Specific aminoacylation by aminoacyl-tRNA synthetases requires accurate recognition of cognate tRNA substrates. In the case of alanyl-tRNA synthetase (AlaRS), RNA duplexes that mimic the acceptor stem of the tRNA are efficient substrates for aminoacylation in vitro. It was previously shown that recognition by AlaRS is severely affected by a simple base pair transversion of the G2:C71 pair at the second position in the RNA helix. In this study, we determined the aminoacylation efficiencies of 50 variants of the tRNA(Ala) acceptor stem containing substitutions at the 2:71 position. We find that there is not a single functional group of the wild-type G2:C71 base pair that is critical for positive recognition. Rather, we observed that base-pair orientation plays an important role in recognition. In particular, pyrimidine2:purine71 combinations generally resulted in decreased aminoacylation efficiency compared to the corresponding purine:pyrimidine pair. Moreover, the activity of a pyrimidine:purine variant could be partially restored by the presence of a major groove amino group at position 71. In an attempt to understand this result further, dielectric continuum electrostatic calculations were carried out, in some cases with additional inclusion of van der Waals interaction energies, to determine interaction potentials of the wild-type duplexAla and seven 2:71 variants. This analysis revealed a positive correlation between major groove negative electrostatic potential in the vicinity of the 3:70 base pair and measured aminoacylation efficiency.     

Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA

Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes. The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule. Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance. Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix. This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region. Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation. Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced. We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed. The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix.     

Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase.

Although the anticodon is the primary element in Escherichia coli tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition. Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAValdemonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency. The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal, to efficient valine acceptors. This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs. Mutational analysis of tRNAValverifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition. Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m. A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix. The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.     

Misacylation of pyrrolysine tRNA in vitro and in vivo

Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.     

Mutant aminoacyl-tRNA synthetase that compensates for a mutation in the major identity determinant of its tRNA

A single G3.U70 base pair in the acceptor helix is the major determinant for the identity of alanine transfer RNAs (Hou & Schimmel, 1988). Introduction of this base pair into foreign tRNA sequences confers alanine acceptance on them. Moreover, small RNA helices with as few as seven base pairs can be aminoacylated with alanine, provided that they encode the critical base pair (Francklyn & Schimmel, 1989). Alteration of G3.U70 to G3.C70 abolishes aminoacylation with alanine in vivo and in vitro. We describe here the mutagenesis and selection of a single point mutation in Escherichia coli Ala-tRNA synthetase that compensates for a G3.C70 mutation in tRNAAla. The mutation maps to a region previously implicated as proximal to the acceptor end of the bound tRNA. In contrast to the wild-type enzyme, the mutant charges small RNA helices that encode a G3.C70 base pair. However, the mutant enzyme retains specificity for alanine tRNA and can serve as the sole source of Ala-tRNA synthetase in vivo. The results demonstrate the capacity of an aminoacyl-tRNA synthetase to compensate through a single amino acid substitution for mutations in the major determinant of its cognate tRNA.     

Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 A resolution.

The acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3.U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase. We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses. The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALA(C70)) determined at 1.4 A exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. We suggest that a highly conserved hydration pattern in both grooves around the G3.U70 wobble base pair may be functionally significant.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Identification of discriminator base atomic groups that modulate the alanine aminoacylation reaction

Specific aminoacylation of tRNAs involves activation of an amino acid with ATP followed by amino acid transfer to the tRNA. Previous work showed that the transfer of alanine from Escherichia coli alanyl-tRNA synthetase to a cognate RNA minihelix involves a transition state sensitive to changes in the tRNA acceptor stem. Specifically, the "discriminator" base at position 73 of minihelix(Ala) is a critical determinant of the transfer step of aminoacylation. This single-stranded nucleotide has previously been shown by solution NMR to be stacked predominantly onto G(1) of the first base pair of the alanine acceptor stem helix. In this work, RNA duplex(Ala) variants were prepared to investigate the role of specific discriminator base atomic groups in aminoacylation catalytic efficiency. Results indicate that the purine structure appears to be important for stabilization of the transition state and that major groove elements are more critical than those located in the minor groove. This result is in accordance with the predicted orientation of a class II synthetase at the end of the acceptor helix. In particular, substitution of the exocyclic amino group of A(73) with a keto-oxygen resulted in negative discrimination at this site. Taken together, these new results are consistent with the involvement of major groove atomic groups of the discriminator base in the formation of the transition state for the amino acid transfer step.     

Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution

We observed recently that a single G3.U70 base pair in the amino acid acceptor stem of an Escherichia coli alanine tRNA is a major determinant for its identity. Inspection of tRNA sequences shows that G3.U70 is unique to alanine in E. coli and is present in eucaryotic cytoplasmic alanine tRNAs. We show here that single nucleotide changes of G3.U70 to A3.U70 or to G3.C70 eliminate in vitro aminoacylation of an insect and of a human alanine tRNA by the respective homologous synthetase. Compared to the influence of G3.U70, other sequence variations in tRNAAla have a relatively small effect on aminoacylation by the insect and human enzymes. In addition, while these eucaryotic tRNAs have nucleotide differences from E. coli alanine tRNA, they are heterologously charged only with alanine when expressed in E. coli. The results indicate a functional role for G3.U70 that is conserved in evolution. They also suggest that the sequence differences between E. coli and the eucaryotic alanine tRNAs at sites other than the conserved G3.U70 do not create major determinants for recognition by any other bacterial enzyme.     

Evidence for interaction of an aminoacyl transfer RNA synthetase with a region important for the identity of its cognate transfer RNA

Recent experiments showed that a single base pair (G3:U70) in the amino acid acceptor helix is a major determinant for the identity of Escherichia coli alanine transfer RNA. Experiments reported here show that bound alanine tRNA synthetase protects (from ribonuclease attack) seven consecutive phosphodiester linkages on the 3'-side of the acceptor-T psi C helix (phosphates 65-71) and a few additional sites that are in scattered locations. There is no evidence for interaction of the enzyme with the anticodon, a sequence which can be varied without effect on recognition by alanine tRNA synthetase.     

Positional recognition of a tRNA determinant dependent on a peptide insertion

The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized. Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position. This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala). Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem. Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate. Larger or smaller deletions were ineffective. The crystal structure of the orthologous A. aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition.     

Evidence for a conserved relationship between an acceptor stem and a tRNA for aminoacylation.

The anticodon-independent aminoacylation of RNA hairpin helices that reconstruct tRNA acceptor stems has been demonstrated for at least 10 aminoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA synthetase, the specificity of aminoacylation of the acceptor stem is determined by the U73 nucleotide adjacent to the amino acid attachment site. Because U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cystein enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelixCys based on the acceptor-T psi C stem of yeast tRNACys is a substrate for the E. coli enzyme, and that aminoacylation of this minihelix is dependent on U73. Additionally, we identify two base pairs in the acceptor stem that quantitatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship between the acceptor stem alone and the acceptor stem in the context of a tRNA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cysteine tRNAs is a tertiary base pair at position 15:48 outside of the acceptor stem. Although E. coli tRNACys has an unusual G15:G48 tertiary base pair, yeast tRNACys has a more common G15:C48 that prevents efficient aminoacylation of yeast tRNACys by the E. coli enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.

The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.