Trans-editing of mischarged tRNAs - PubMed (original) (raw)

Trans-editing of mischarged tRNAs

Ivan Ahel et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs. Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3' ends of tRNAs. Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cis-editing). Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme. A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNAPro. Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze Ser-tRNAAla and Gly-tRNAAla substrates. The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Multiple alignment of the ProRS editing domain homologues. Asterisks represent residues that were shown important for editing function of the E. coli ProRS enzyme. “>OXH<” defines the position of the oxyanion hole. The last two sequences represent Ybak proteins [the crystal structure has been solved for the H. influenzae protein (17)]. M.mus, Mus musculus (NM_026465); M.mag, Magnetospirillum magnetotacticum (ZP_00053959); R.rub, Rhodospirillum rubrum (ZP_00016210); C.sti, C. sticklandii (CAB71308); P.fal, P. falciparum (AE014846); T.bru, Trypanosoma brucei (

www.sanger.ac.uk/cgi-bin/blast/getseq?id=115XH77A6700k03xeEJ;db=tryppub/contigs;acc=tryp\_x-97f05.q1k

); E.col, E. coli (ProRS AAB08622, Ybak AAC73583); B.sub, Bacillus subtilis (NP_389539); C.ace, Clostridium acetobutylicum (NP_349775); A.aeo, Aquifex aeolicus (NP_213250); H.inf, H. influenzae (P45202); Ins, insertion domain of bacterial-type ProRS; Nterm, N-terminal extension in ProRSs from lower eukaryotes. GenBank accession numbers are in parentheses.

Fig. 2.

Fig. 2.

Different architectures of the Prolyl-tRNA synthetases and their editing domains. In blue are shown editing domains (note that they can be present in cis or trans; as an N-terminal domain or an insertion). In red are shown catalytic domains (orange is used for bacterial-type ProRS). Anticodon-binding domains are yellow, and archaeal-type ProRS-specific C termini are green. Representatives of the analyzed editing domain homologues are shown in parentheses.

Fig. 3.

Fig. 3.

Unrooted phylogenetic tree implied by the neighbor-joining method showing possible relationships between ProRS insertion domain homologues. If not specifically labeled in genomic databases, the free-standing ProRS editing domain homologues are denoted ProX. GenBank accession numbers are in parentheses: C. sticklandii PrdX (CAB71308), Pseudomonas aeruginosa ProX (AAG05230), M. magnetotacticum ProX (ZP_00053959), Agrobacterium tumefaciens ProX1 (NP_356919), M. musculus ProX (NM_026465), Arabidopsis thaliana ProX (AAO63892), P. falciparum ProRS (AE014846), Chlamydia trachomatis ProRS (NP_219903), E. coli ProRS (AAB08622), Thermotoga maritima ProRS (NP_228324), C. acetobutylicum ProRS (NP_349775), E. coli YeaK (AAC74857), Chlorobium tepidum ProX (NP_662760), Aeropyrum pernix ProX (NP_148681), A. tumefaciens ProX2 (AAK85924), Enterococcus faecalis EbsC (AAC36853), Streptomyces coelicolor ProX (NP_626318), E. coli YbaK (AAC73583), and H. influenzae YbaK (P45202).

Fig. 4.

Fig. 4.

Multiple alignments of the editing modules of AlaRS and ThrRS. Asterisks represent residues that were shown to be important for the editing function of the E. coli ThrRS or the E. coli AlaRS enzymes. M.jan, M. jannaschii (Q57984); P.aby, Pyrococcus abyssi (Q9UY36); E.col, E. coli (Q8XE27); B.sub, B. subtilis (P18256); M.bar, M. barkeri (ZP_00076591); A.per, A. pernix (NP_147589); S.sol, S. solfataricus (AlaX1, NP_341986; AlaX2, NP_342718); D.rad, Deinococcus radiodurans (NP_294225). GenBank accession numbers are in parentheses. S. solfataricus possesses two different AlaX.

Fig. 5.

Fig. 5.

Unrooted phylogenetic tree implied by the neighbor-joining method, showing relationships between the editing domains of AlaRS and ThrRS and their free-standing homologues found elsewhere. Note that the homologous module is found as an insert (AlaRS), an N-terminal extension (ThrRS), and a freestanding protein (AlaX). A homologous domain was also found at the N terminus of Plasmodium TrpRS. GenBank accession numbers are in parentheses: D. radiodurans AlaX (NP_294225), M. barkeri AlaX (ZP_00076591), S. solfataricus AlaX1 (NP_341986), P. aeruginosa AlaX (NP_250796), Mesorhizobium loti AlaX (NP_102167), D. radiodurans AlaRS (Q9RS27), C. acetobutylicum AlaRS (Q97IG3), Giardia lamblia AlaRS (AAG23137), M. barkeri AlaRS (ZP_00077929), M. jannaschii AlaRS (Q57984), Thermoplasma volcanium ThrRS (Q978W0), Bacillus halodurans ThrRS (Q9K866), T. maritima ThrRS (Q9WZJ9), Rickettsia prowazekii ThrRS (O05947), E. coli ThrRS (Q8XE27), P. falciparum TrpRS (NP_705269), Dictyostelium discoideum AlaX (AAO52108), Pyrococcus horikoshii AlaX2 (NP_142539), S. solfataricus AlaX2 (NP_342718), Homo sapiens AlaX (NP_079543), S. cerevisiae AlaX (NP_014358).

Fig. 6.

Fig. 6.

(A) Specific deacylation of E. coli Ala-tRNAPro. Shown are 0.3 μM CSPrdX (•), 0.3 μM CSPrdX plus 1 μM CSProRS (▴), 0.3 μM ECYbaK (▪), 0.5 μM PFProRS (○), 0.5 μM ΔPFProRS (□), 0.5 μM S. cerevisiae ProRS (▵), 0.1 μM E. coli ProRS (▾), and control without enzyme (▿). (Inset) Pro-tRNAPro and Ala-tRNAAla deacylation [0.3 μM CSPrdX (• and ○, respectively), 0.5 μM PFProRS (▪ and □, respectively)]. (B) Mischarging of alanine onto S. cerevisiae tRNAPro. Shown are 6 μM PFProRS (•), 6 μM ΔPFProRS (▪), and 6 μM S. cerevisiae ProRS (▵).

Fig. 7.

Fig. 7.

Specific deacylations by AlaXs. Shown are 100 nM M. barkeri AlaX and Ser-tRNAAla (•), 100 nM S. solfataricus AlaX and Ser-tRNAAla (▪), 100 nM M. jannaschii AlaRS and Ser-tRNAAla (▴), 100 nM S. cerevisiae AlaX and Ser-tRNAAla (□), 100 nM M. barkeri AlaX and Gly-tRNAAla (○), 100 nM M. barkeri AlaX and Ser-tRNAThr (▾), and control without enzyme (▵).

Similar articles

Cited by

References

    1. Fersht, A. R. (1981) Proc. R. Soc. London Ser. B. 212, 351–379. - PubMed
    1. Jakubowski, H. & Goldman, E. (1992) Microbiol. Rev. 56, 412–429. - PMC - PubMed
    1. Giegé, R., Sissler, M. & Florentz, C. (1998) Nucleic Acids Res. 26, 5017–5035. - PMC - PubMed
    1. Jakubowski, H. & Fersht, A. R. (1981) Nucleic Acids Res. 9, 3105–3117. - PMC - PubMed
    1. Beebe, K., Ribas De Pouplana, L. & Schimmel, P. (2003) EMBO J. 22, 668–675. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources