The ribosome requires a full complement of correctlyaminoacylated tRNAs in order to perform mRNA-templated protein synthesis. The aminoacyl-tRNA synthetase (AARS) family of proteins comprises the principal catalysts of aminoacyl-tRNA (AA-tRNA) formation.The ability of an AARS to discriminate between multiplesubstrates and to accurately attach a particular amino acidto its cognate tRNA species is a universal requirement forthe faithful translation of genetic information into the sequence of a polypeptide. In the pregenomic era it was agenerally held belief, as originally propounded in theadaptor hypothesis (Crick 1958), that virtually every celland organelle would contain 20 AARSs, with one particular enzyme responsible for the synthesis of each of the20 AA-tRNA isoacceptor species required for mRNAtranslation. The identification in Escherichia coli of twodistinct AARS classes, each containing 10 members, provided seemingly incontrovertible support for the "20AARS" facet of the adaptor hypothesis. This view of AAtRNA synthesis was initially confounded when thegenomes of two methanogenic archaea were sequencedand annotated (Bult et al. 1996; Smith et al. 1997). A variety of analyses consistently identified genes encodingonly 16 AARSs, raising the question of how these organisms synthesize asparaginyl tRNA (Asn-tRNA), cysteinyl tRNA (Cys-tRNA), glutaminyl tRNA (Gln-tRNA),and lysyl tRNA (Lys-tRNA). The identification of the alternative pathways by which these four AA-tRNAs aremade has provided unexpected insight into the structuraland functional diversity of AA-tRNA synthesis not envisaged within the structures of the 20 AARS hypothesis.Furthermore, functional genomics and comparative phylogenetic studies have shown that, far from beingidiosyncrasies of methanogenic archaea, these alternativepathways of AA-tRNA synthesis are widespread in theliving kingdom. To date, three novel activities have beencharacterized: an unusual class I lysyl-tRNA synthetasefor Lys-tRNA formation (Ibba et al. 1997c), tRNA-dependent amidation enzymes for Asn-tRNA and GlntRNA synthesis (Tumbula et al. 2000), and a dual-specificity prolyl-tRNA synthetase generating Cys-tRNA andPro-tRNA (Stathopoulos et al. 2000). Here we describethe current state of knowledge on these unusual routes forAA-tRNA synthesis...

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes. To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined. Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an Escherichia coli strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS in vivo. In vitro characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases. Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNALys as their class II counterparts, namely the discriminator base (N73) and the anticodon. This sequence-specific recognition of the same nucleotides in tRNALys by the two unrelated types of enzyme suggests that tRNALys predates at least one of the LysRSs in the evolution of the translational apparatus. The only observed variation in recognition was that the G2.U71 wobble pair of spirochete tRNALys acts as antideterminant for class II LysRS but does not alter class I enzyme recognition. This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNALys species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria.

Lysyl-tRNA synthetases are unique amongst the aminoacyl-tRNA synthetases in being composed of two unrelated families. In most bacteria and all eukarya, the known lysyl-tRNA synthetases are subclass nc-type aminoacyl-tRNA synthetases whereas some archaea and bacteria have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. We have now examined substrate recognition by a bacterial (from Borrelia burgdorferi) and an archaeal (from Methanococcus maripaludis) class I lysyl-tRNA synthetase. The genes encoding both enzymes were able to rescue an Escherichia coli strain deficient in lysyl-tRNA synthetase, indicating their ability to functionally substitute for class II lysyl-tRNA synthetases in vivo. In vitro characterization revealed lysine activation and recognition to be tRNA-dependent, a phenomenon previously reported for other class I aminoacyl-tRNA synthetases. More detailed examination of tRNA recognition has shown that class I lysyl-tRNA synthetases recognize the same elements in tRNA(Lys) their class II counterparts; specifically, the discriminator base (N73) and the anticodon serve as recognition elements. The implications of these results for the evolution of Lys-tRNA(Lys) synthesis and their possible indications of a more ancient origin for tRNA then aminoacyl-tRNA synthetases will be discussed.