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One Polypeptide with Two Aminoacyl-tRNA Synthetase Activities

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Science  21 Jan 2000:
Vol. 287, Issue 5452, pp. 479-482
DOI: 10.1126/science.287.5452.479

Abstract

The genome sequences of certain archaea do not contain recognizable cysteinyl–transfer RNA (tRNA) synthetases, which are essential for messenger RNA–encoded protein synthesis. However, a single cysteinyl–tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl–tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl–tRNA synthetase can synthesize both cysteinyl-tRNACys and prolyl-tRNAPro. The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.

The insertion of cysteine into nascent peptides during protein synthesis is dependent on the interaction of cysteine codons with cysteinyl-tRNA (Cys-tRNA) in the ribosomal A site. Cys-tRNA is synthesized from cysteine and tRNACys in an adenosine 5′-triphosphate (ATP)–dependent reaction catalyzed by cysteinyl–tRNA synthetase (CysRS). Genes encoding CysRS, cysS, have been detected in over 40 organisms encompassing all the living kingdoms (1). The only known exceptions are the thermophilic methanogensMethanococcus jannaschii and Methanobacterium thermoautotrophicum, the complete genome sequences of which contain no open reading frames encoding cysS homologs (2), raising the question of how these archaea synthesize Cys-tRNA for protein synthesis. It was initially suggested that Cys-tRNACys might be synthesized by a pathway involving modification of a mischarged tRNA such as Ser-tRNACys, using a mechanism reminiscent of those previously described for the synthesis of asparaginyl-tRNA (Asn-tRNA), glutaminyl-tRNA (Gln-tRNA) and, more specifically, selenocysteinyl-tRNA (Sec-tRNA) (3). Biochemical analyses revealed no evidence for such a pathway (4) but instead showed that Cys-tRNA is synthesized directly from cysteine and tRNA in an ATP-dependent reaction (5). The identity of the enzyme responsible for Cys-tRNA synthesis in M. jannaschii andM. thermoautotrophicum is unknown. The recent finding that some aminoacyl–tRNA synthetase (AARS)–encoding genes may be dispensable for cell viability also raised the possibility thatcysS genes might be absent altogether from the genomes ofM. jannaschii and M. thermoautotrophicum(6).

To investigate how Cys-tRNA is synthesized in M. jannaschii, we attempted to purify from cell-free extracts a protein with CysRS activity. Conventional chromatographic procedures (7) led to the isolation of a single protein with normal CysRS activity (Fig. 1, A and B). Protein analysis revealed an 18–amino acid peptide sequence matching the predicted NH2-terminal sequence of M. jannaschiiprolyl–tRNA synthetase (ProRS) (Fig. 1C). A test of this protein with CysRS activity confirmed that it also possessed ProRS activity. This suggested that the determined sequence might arise from a contaminating protein rather than from the bona fide CysRS. To address this question, we cloned the gene encoding M. jannaschiiProRS (proS) and used it for heterologous expression in Escherichia coli and subsequent purification of ProRS (8). In vitro aminoacylation assays showed thatM. jannaschii ProRS could synthesize both Cys-tRNA and Pro-tRNA at comparable rates (Fig. 1, D and E), suggesting dual amino acid specificity for this enzyme during protein synthesis. Prolonged aminoacylation showed that the M. jannaschii enzyme could generate 68 pmol of Cys-tRNA per A 260(absorbance at 260 nm) unit of unfractionated homologous tRNA (pure tRNA species accept ∼1600 pmol perA 260 unit), a much higher level (4.3%) than observed before (5) and consistent with the tRNACys content in the tRNA of other organisms. Similarly, the enzyme was efficient in proline charging (78 pmol perA 260 unit).

Figure 1

Purification and NH2-terminal sequencing of a protein with CysRS activity from M. jannaschii. Protein purification was monitored by SDS-PAGE (molecular size standards are in kD). (A) Active eluate from the final chromatographic step (Sepharose 4B-CNBr activated with 100 mg of total E. coli tRNA as a coupling ligand). (B) Samples (20 μl) of active fractions from the previous step were loaded on a 10 to 20% gradient tris-glycine native gel and subjected to PAGE. The bands were located by staining of part of the gel with Coomassie Blue, then were excised from the gels. After overnight elution in reaction buffer, CysRS activity was tested. The boxed band corresponds to the only sample that contained CysRS activity. (C) The active band from (B) was further analyzed by SDS-PAGE, blotted onto polyvinylidene difluoride membrane, and subjected to NH2-terminal sequencing. The 18–amino acid sequence derived (shown) corresponds to ProRS from M. jannaschii. This enzyme was then heterologously produced inE. coli and purified and tested for both CysRS and ProRS activities. M. jannaschii ProRS was found to catalyze the direct attachment of both cysteine (D) and proline (E) to a fraction of M. jannaschii total tRNA. Aminoacylation reactions (20-μl samples) were performed as described in the presence of the following amino acids: (D) 20 μM [3H]proline (•); 20 μM [3H]proline and 800 μM nonradiolabeled cysteine (▴); and 20 μM [3H]proline and 800 μM nonradiolabeled proline (○). (E) Same as (D), but with 20 μM [35S]cysteine instead of 20 μM [3H]proline. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; I, Ile; K, Lys; L, Leu; M, Met; S, Ser; W, Trp; and Y, Tyr.

The possibility that the observed CysRS-like activity results from the mischarging of tRNAPro with Cys to yield Cys-tRNAPro was investigated. A transcript of the M. jannaschii tRNAPro gene was synthesized in vitro and then purified. Attempts to aminoacylate this tRNA transcript with M. jannaschii ProRS showed that it can readily be charged with Pro but not with Cys (9). Additionally, unfractionated M. jannaschii tRNA was charged with Pro and subsequently treated with sodium metaperiodate which oxidized, and thus inactivated, all uncharged tRNAs (10). After deacylating the Pro-tRNA, we attempted to recharge the tRNA preparation; although Pro charging activity was still detectable, Cys charging had now been abolished, indicating that Pro was exclusively attached to tRNAPro in the initial reaction (11).Methanococcus jannaschii total tRNA was also partially fractionated into its various isoacceptors by reversed-phase liquid chromatography, and these fractions were subsequently tested for their ability to be charged with Cys and Pro by M. jannaschiiProRS (12). A single fraction solely chargeable with Cys and two discrete fractions solely chargeable with Pro were detected, in agreement with the prediction from the genome sequence that M. jannaschii contains one tRNACysand two tRNAPro isoacceptors. These data indicate that M. jannaschii ProRS is able to synthesize both Cys-tRNACys and Pro-tRNAPro, but not Cys-tRNAPro or Pro-tRNACys.

To examine the ability of M. jannaschii ProRS to synthesize Cys-tRNA in vivo, we attempted to rescue growth at a restrictive temperature of an E. coli cysS temperature-sensitive mutant strain using the archaeal proS gene (13). Coexpression of the genes encoding M. jannaschiitRNACys and various methanogen ProRS proteins restored growth of E. coli UQ818 at 42°C, indicating that ProRS can synthesize Cys-tRNACys in vivo (Fig. 2). The slow growth of the rescued transformants was attributed to the high number of rare codons (with respect to normalE. coli usage; e.g., for arginine) in the archaeal genes, and perhaps also to an unfavorable cellular Cys:Pro ratio. In addition, the apparent need for modification of tRNACys to render it active (evidenced by the inactivity of a gene transcript) suggests that the tRNA substrate may be less than optimal when expressed in E. coli. In vivo complementation was strictly dependent on the presence of the M. jannaschiitRNACys gene, indicating the archaeal proSgene products could not charge E. colitRNACys sufficiently to sustain growth. The ability of M. jannaschii ProRS to synthesize Cys-tRNA both in vitro and in vivo, together with the lack of a cysS gene in the genome of M. jannaschii, indicates that this enzyme can specify two amino acids during protein synthesis.

Figure 2

Complementation of a temperature-sensitivecysS mutation in E. coli strain UQ818 withproS genes of M. maripaludis, M. jannaschii, M. thermoautotrophicum, and acysS gene from E. coli. The experiment was performed as described (11). An additional plasmid (pTech-Mj-tRNACys) containing the M. jannaschii tRNACys gene was necessary in strain UQ818 (see text). The plates were incubated for 4 days at the permissive temperature (30°C) (A) or at the nonpermissive temperature (42°C) (B).

The observation that M. jannaschii ProRS can recognize both Cys and Pro raised the question of how such recognition is achieved in the context of a single protein. Synthesis of [35S]Cys-tRNA and [3H]Pro-tRNA were both inhibited by the addition of excess unlabeled Cys or Pro (Fig. 3, A and B) and by the ProRS inhibitor thiaproline (14) (Fig. 3C). Thus, either the active center of ProRS contains both Cys and Pro binding sites in close proximity, or the protein contains two functionally linked active sites. Another possible explanation is that ProRS displays a broad range of amino acid specificity under the in vitro experimental conditions used, as recently described for E. colilysyl–tRNA synthetase (15). However, the inability of any of the other 18 canonical amino acids to inhibit aminoacylation by ProRS (Fig. 4, A and B) indicates that binding is specific for Cys and Pro. In addition, Cys (but not Pro) activation was found to require the presence of tRNA (Fig. 4C), indicating that in vivo there are effectively two separate entities, a free ProRS that recognizes proline and a ProRS:tRNACys complex that recognizes cysteine.

Figure 3

Aminoacylation of M. jannaschii tRNA by purified M. jannaschii His6-ProRS. Aminoacylation reactions (20-μl samples) were performed as described in the presence of the following amino acids: (A) 20 μM [35S]cysteine (•); 20 μM [35S]cysteine and 800 μM nonradiolabeled cysteine (▴) and 20 μM [35S]cysteine and 800 μM nonradiolabeled proline (○). (B) Same as (A), but with 20 μM [3H]proline. (C) Inactivation of the formation of Cys-tRNACys (•) and Pro-tRNAPro (▪) by 800 μM thiaproline (open squares and open circles, respectively).

Figure 4

Specific activation of Cys and Pro by M. jannaschii His6-ProRS. Reactions were performed as described (A) in the presence of 20 μM [35S]cysteine (•), 20 μM [35S]cysteine and the 18 nonradiolabeled amino acids (800 μM), with the exception of cysteine and proline (□), and 20 μM [35S]cysteine and 800 μM nonradiolabeled cysteine (▴) or proline (○). (B) The same as in (A) but with 20 μM [3H]proline instead of 20 μM [35S]cysteine. (C) Cysteine-dependent pyrophosphate exchange. Activation of cysteine by M. jannaschii ProRS was observed only in the presence of totalM. jannaschii tRNA (1 μg/μl) (•). In the conditions used for the reaction {2 mM cysteine, 1 mM [32P]PPi (NEN DuPont, 4.6 Ci/mmol)}, no amino acid activation was observed in the presence of in vitro–transcribed tRNAPro (▾), in vitro–transcribed tRNACys (○), with fractionated M. jannaschii tRNA lacking tRNACys (▴), or in the presence of the enzyme alone (□).

The finding that M. jannaschii ProRS also functions as a CysRS is unexpected. Normally, individual aminoacyl-tRNAs are synthesized by a particular AARS specific for the appropriate amino acid and tRNA, with errors in substrate recognition being corrected by proofreading (16). The only known exceptions are Asn-tRNA, Gln-tRNA, and Sec-tRNA, which can be synthesized by reaction schemes dependent on the initial recognition of apparently noncognate tRNAs by AARSs, although amino acid recognition by the appropriate enzyme remains specific (3). The CysRS activity of M. jannaschii ProRS differs in that it is dependent on the enzyme using both Cys and tRNACys as cognate substrates.

The amino acid sequences of the M. jannaschii and M. thermoautotrophicum ProRSs show a high degree of similarity to the sequences of other ProRS proteins (17), indicating that any differences associated with their CysRS function cannot be detected by phylogenetic methods alone (18). Furthermore, CysRS activity may exist in ProRS enzymes from organisms with a conventional cysS gene, in which case the M. jannaschii and M. thermoautotrophicum ProRSs would not be expected to be distinctive in amino acid sequence. This is supported by the observation that Methanococcus maripaludis contains both a ProRS with CysRS activity (Fig. 2) and a CysRS (1). It raises the question of whether the CysRS synthetic activity is an ancestral feature or has been more recently acquired by ProRS to compensate for the loss of a conventional cysS gene. The detection of vestigial thiol-binding sites in other class II aminoacyl–tRNA synthetases (19) suggests that Cys-tRNA synthetic activity could have evolved in such enzymes. A sampling of other ProRS proteins to delineate the distribution of CysRS activity is now needed to address the evolutionary timing of such an event and how it might relate to the distribution of cysS genes.

The existence of AARSs able to catalyze the synthesis of more than one aminoacyl-tRNA is assumed to have been an important step in the evolution of these enzymes (20). This is at odds with the narrow substrate ranges seen in contemporary AARSs, a characteristic critical for their role in translation. However, the ability of ProRS to synthesize two aminoacyl-tRNAs suggests that the AARSs could have evolved via ancestors characterized by wide substrate specificities. The fact that most organisms now contain separate AARSs for the synthesis of each aminoacyl-tRNA, rather than a limited number of enzymes with multiple activities, suggests that functional isolation of these pathways offers a selective advantage. Such an advantage may be related to the known ability of individual aminoacyl-tRNA and AARS levels to fine tune the expression of AARS-encoding genes, thus providing a means to more precisely regulate individual components of the translational machinery (21).

Numerous schemes have been proposed for the evolution of translation (22), many of which suggest that early protein synthesis was a relatively unspecific process giving rise to mixed populations of polypeptides [e.g., (23)]. Within such schemes, amino acid activation is assumed to have been achieved by ancestral AARS-like enzymes able to recognize a broad range of amino acids (24). The evolution of the extant AARS proteins from such precursors would require intermediates with multiple substrate specificities, an activity now shown to exist also in a contemporary AARS, M. jannaschii prolyl–tRNA synthetase.

  • * To whom correspondence should be addressed at the Department of Molecular Biophysics and Biochemistry, Yale University, Post Office Box 208114, 266 Whitney Avenue, New Haven, CT 06520–8114, USA. E-mail: soll{at}trna.chem.yale.edu

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