Discrimination between transfer-RNAs by tyrosyl-tRNA synthetase

tRNA determinants for transcription antitermination of the Bacillus subtilis tyrS gene

“…The composite (COMP) tRNA Tyr construct generated from the intermediate constructs contained all of the features of a normal tRNA but had several primary sequence differences relative to tRNA Tyr (Fig+ 2C); none of these changes are predicted to disrupt known tertiary interactions (Giege et al+, 1993)+ Expression of tRNA Tyr COMP yielded 76 units of b-galactosidase expression from the tyrS-lacZ AMB-U222A fusion, in comparison to 1+5 units of expression in the absence of an appropriate tRNA Tyr variant (Table 2)+ This level of tyrS readthrough was identical to that obtained with an equivalent construct containing tRNA Tyr AMB-A73U, indicating that the stem region substitutions introduced did not negatively affect readthrough+ A second variant (BGII; Fig+ 2D) was generated that contained an additional restriction endonuclease cleavage site in the D stem and consequently an extra base pair in the D stem (C13-G22), at a position that also participates in an interaction with U47+ The BGII variant also contained a substitution of the 9{23{12 interaction of normal tRNA Tyr (A{C{G) with A{U{U+ In addition, position 11, which is a pyrimidine in nearly all bacterial tRNAs except tRNA fmet (Sprinzl et al+, 1998), was replaced with a purine+ This variant resulted in a reduction of tyrS-lacZ fusion expression to 20 units, suggesting that Tyr was placed at the ϩ1 position of the rpsD promoter using an XmaI restriction site; the 39 position of tRNA Tyr was placed immediately upstream of the 59 position of a variant of tRNA Gln using an NcoI site, so that RNase P cleavage directed by the downstream tRNA Gln would simultaneously release the mature 39 end of tRNA Tyr + The NcoI site contains the CCA that is common to all tRNA 39 ends+ In addition to the XmaI and NcoI sites, Site 1 and Site 2 represent unique restriction sites placed at various positions of the tRNA, permitting replacement of the region between the sites by oligonucleotide cassettes containing nucleotide substitutions at defined positions+ B: MINI: contains the acceptor and anticodon arms+ C: MINI ϩ D: contains the acceptor, anticodon, and D arms+ D: MINI ϩ TV: contains the acceptor, anticodon, T, and variable arms+ the D stem sequence can influence the system, although activity was still high+ Nonsense suppression by the tRNA variants was assessed as an indicator of charging efficiency (Table 2)+ The wild-type tRNA Tyr AMB-A73U construct conferred 25 units of b-galactosidase expression using the rpsD Am -lacZ translational fusion, somewhat higher than the 13 units obtained with Pspac-dependent expression (Table 1); this increase is presumably due to the increased level of tRNA expression directed by the stronger rpsD promoter (F+ Grundy & T+ Henkin, unpubl+ results)+ The tRNA Tyr COMP variant gave a threefold increase in amber suppression relative to wild-type tRNA Tyr AMB-A73U, suggesting that the stem sequence alterations may enhance charging efficiency; because TyrRS does not recognize tRNAs with the A73U substitution (Bedouelle et al+, 1993), another aminoacyl-tRNA synthetase may be responsible for this activity+ In contrast, the D stem changes of the BGII construct, while resulting in a 3+8-fold decrease in tyrS-lacZ expression, dropped amber suppression to background levels (150-fold reduction relative to the COMP variant), indicating a severe defect in tRNA charging+ This construct was therefore used for most further mutagenesis experiments, to eliminate possible tRNA charging variability+…”

“…As a first step in determining the flexibility of the structural requirements for tRNA Tyr for tyrS antitermination, E. coli tRNA Tyr was introduced into B. subtilis+ E. coli tRNA Tyr contains multiple differences at the primary sequence level from its B. subtilis homolog (Fig+ 2)+ These differences include some of the key tertiary interaction positions (Giege et al+, 1993); although the 15{48 trans interaction is maintained, the 26{44 cis interaction (G{U) Tyr is known to interact with the leader by pairing of the anticodon (CUA, outlined letters) with the specifier sequence (boxed UAG amber codon) and by pairing of the 39 acceptor end of the tRNA (UCCA, outlined letters) with the side bulge of the antiterminator (UGGA)+ The remainder of the tyrS leader is omitted for clarity (dashed lines)+ Numbering of the tyrS leader is relative to the start point of transcription (Henkin et al+, 1992)+ is replaced by A{C, the (47{45)(10{25) interaction [(U{C)(G-C)] is replaced by (U{G)(C-G), and the (13{22){47 interaction [(A{A){U] is replaced by (G{A){U in E. coli tRNA Tyr + The supF amber suppressor allele of tRNA Tyr , containing a CUA anticodon to match UAG amber codons (Kirsebom & Svard, 1992), was modified by introduction of an A73U mutation at the discriminator position of the tRNA to reduce possible interference by charged tRNA+ This substitution blocks tRNA Tyr charging by TyrRS (Bedouelle et al+, 1993), and it was previously demonstrated that this mutation in B. subtilis tRNA Tyr AMB, a supF analog, reduced amber suppression in vivo and permitted induction of a tyrS-lacZ fusion with a corresponding U222A substitution at the antiterminator and UAG specifier sequence during growth in rich medium (Grundy et al+, 1994)+ The mutant tRNA was inserted into plasmid pDG148 (Stragier et al+, 1988) so that expression would be directed by the IPTG-inducible Pspac promoter+ This construct was introduced into B. subtilis tester strains containing single-copy tyrS-lacZ fusions integrated into an SPb prophage+ Expression of the tyrS-lacZ AMB-U222A fusion in the absence of plasmid-directed tRNA synthesis was low, and IPTG-induced synthesis of tRNAs lacking a match at the specifier (pWT) or at the U222A position (pAMB) failed to significantly increase expression of the fusion (Table 1 Am -lacZ translational fusion, which contains an amber mutation in the ribosomal protein S4 coding sequence fused to a lacZ reporter (Table 1); this fusion was previously used to assay translational suppression (Grundy & Henkin, 1994b) An expression system that eliminated the requirement for the tRNA to provide the normal processing determinants was developed to facilitate generation of large numbers of variants of tRNA Tyr , some of which could be expected to alter these determinants+ The expression cassette (Fig+ 3) placed the first position of the tRNA (using an XmaI restriction site) at the transcription start point of the very strong B. subtilis rpsD promoter (Grundy & Henkin, 1990), so that the primary transcript would contain the mature 59 end of the tRNA (with a 59 triphosphate, rather than the monophosphate normally generated by RNaseP cleavage of a longer precursor tRNA)+ The 39 terminal A of the tRNA was placed immediately upstream of the 59 position of a variant of tRNA Gln , so that RNaseP cleavage at the 59 end of tRNA Gln , directed by tRNA Gln , would simultaneously lib...…”

“…The sequence and structural requirements for tRNA Tyr -directed antitermination of the B. subtilis tyrS leader were analyzed using a system in which tRNA expression in vivo was separated from other tRNA functions+ This study revealed that although many substitutions were tolerated, especially within helical regions of the tRNA, virtually all of the elements normally conserved in tRNA were required for productive interaction with the tyrS leader+ Substitutions in sequence elements known to be important for formation of the tRNA tertiary structure resulted in loss of tyrS antitermination, indicating that the interaction between the leader and the tRNA is likely to involve recognition of the overall tertiary structure of the tRNA rather than only basepairing interactions+ In all positions of the tRNA where certain residues are conserved in all tRNA species, only the normal residues were permitted; in some cases, such as the T loop, the constraints for tRNA Tyr function in antitermination appeared to exceed the stringency of the rules as established by sequence conservation+ It is likely that different members of the T box system may vary in the details of the tRNA-leader RNA interaction, because each leader has probably evolved to permit efficient interaction with its cognate tRNA and discrimination against noncognate tRNA species; these constraints are analogous to those required for specific tRNA recognition by aminoacyl-tRNA synthetases+ Recognition of tRNAs by aminoacyl-tRNA synthetases in many cases involves both sequence and structural elements (Sampson et al+, 1990;Bedouelle et al+, 1993;Giege et al+, 1993;Puglisi et al+, 1993;McClain et al+, 1999)+ It is therefore not surprising that the tRNA-leader interaction is similarly constrained, although the requirements for aminoacylation and antitermination are different+ Although much of the specificity for antitermination is derived from the specifier-anticodon pairing, as well as the pairing of the variable position of the antiterminator with the discriminator position of the tRNA, it is apparent from both leader and tRNA mutant analyses that other elements participate in this recognition process+…”

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

“…Notably, nine of the fifteen amino acids involved in stabilizing the transition state for the first step of the reaction in the B. stearothermophilus enzyme (11,40,41) are conserved in the human, M. jannaschii, and S. cerevisiae enzymes. In contrast, none of the eleven amino acids known to be involved in tRNA Tyr recognition (42)(43)(44)(45)(46)(47) are conserved between the human and B. stearothermophilus tyrosyl-tRNA synthetases, suggesting that in contrast to the mechanism for formation of the E⅐Tyr-AMP intermediate, tRNA Tyr recognition differs between eukaryotes, archaea, and bacteria. This is most apparent in the M. jannaschii amino acid sequence, which surprisingly is missing a substantial portion of the tRNA Tyr anticodon recognition domain (amino acids 330 -419 in the B. stearothermophilus enzyme).…”

Human mitochondrial TyrRS disobeys the tyrosine identity rules

“…This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNA Tyr Tyrosyl-tRNA synthetase (TyrRS) is distinguished from other aminoacyl-tRNA synthetases by its ambiguous assignation to class I of synthetases (Bedouelle 2004). Indeed, this homodimeric enzyme with typical class I motifs binds its homologous tRNA Tyr across its inferface (Bedouelle and Winter 1986) via the major groove side of the amino acid acceptor stem, as occurs for class II synthetases (Lee and RajBhandary 1991;Bedouelle et al 1993;Fechter et al 2000;Yaremchuk et al 2002). Furthermore, tyrosylation reactions are peculiar in that no cross-aminoacylation between eubacterial tRNA Tyr and eukaryal TyrRS is possible and viceversa (Kleeman et al 1997;Wakasugi et al 1998).…”