Operational RNA code for amino acids: species-specific aminoacylation of minihelices switched by a single nucleotide.

Abstract

The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodonindependent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a speciesspecific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals.The 20 synthetases are divided into two classes (I and II) of 10 enzymes each (1, 2). Enzymes of each class are approximately comprised of two major domains, where each of the two tRNA domains interacts with a distinct domain of the cognate tRNA synthetase (3-5). The tRNA acceptor-TTC helix ("minihelix" domain) interacts with the class-defining catalytic domain (and insertions into that domain), and segments outside of the acceptor-TTC stem-loop, such as the anticodon, interact with a second, highly variable synthetase domain that is joined to the catalytic core. While many synthetases make contact with their tRNA anticodons, specific mutations in the acceptor stems of their tRNAs severely reduce aminoacylation efficiency (6-8) and, even for these "anticodon" examples, RNA oligonucleotides with sequences based on acceptor stems alone are also aminoacylated with their cognate amino acids (9-11). The operational RNA code based on acceptor stems may have predated the genetic code and was possibly incorporated into or combined with the genetic code when the two domains of tRNAs were assembled into a single molecule (4).Escherichia coli glycyl-tRNA synthetase has an a212 quaternary structure, with a 303-amino acid a chain and 689-amino acid 13 subunit (12, 13). In contrast, the human enzyme is an a2 dimer of 739-amino acid polypeptides (14, 15), similarThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be her...

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

“…These results are in good agreement with previously published comparisons between bacterial and eukaryotic tyrosyl-tRNA synthetases (17)(18)(19)(20)(21). Similar species specificity has also been observed for several other aminoacyl-tRNA synthetases (55)(56)(57)(58)(59) and is consistent with the hypothesis that the recognition of tRNA by aminoacyl-tRNA synthetases was still evolving after the divergence of bacteria and eukaryotes (39). The observation that tyrosyl-tRNA synthetase from the archaeon M. jannaschii lacks a substantial portion of the tRNA anticodon recognition domain suggests that the archaea may recognize and bind tRNA Tyr in a manner distinct from that found in both bacteria and eukaryotes.…”

tRNA anticodon shifts in eukaryotic genomes

“…It is possible in vitro to alter a tRNA's amino acid charging identity by anticodon mutations (Schulman and Pelka 1989;Pallanck and Schulman 1991). It has also been shown that mutations outside the anticodon, for example, involving acceptor stem nucleotides that are strong identity determinants, can also effectively switch the identity of a tRNA (Hipps et al 1995). That "identity switches" could potentially occur by single or a few mutations was first demonstrated in vivo by the rescue of an inviable tRNA-Thr(UGU) deletion mutant of E. coli by a mutant tRNA-Arg(UGU) gene (Saks et al 1998).…”

“…It is known that some tRNAs have different identity rules in different taxa. For example Escherichia coli tRNA-Gly uses a crucial U73 discriminator base in combination with a C2:G71 pair, while in mammals the discriminator base is A73 (Shiba et al 1994;Hipps et al 1995); prokaryotic tRNA-Asp uses a U73 and the first base pair G1:C72 of the stem, while eukaryotes use A73 and the third base pair of the stem (Nameki et al 1992. Although crucial to our understanding of the evolution of the genetic code (Schimmel et al 1993), the evolution of tRNA identity elements has been the subject of few studies.…”

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

“…We also considered that the evolution of a synthetase is codeveloped with the evolution of tRNA determinants that are necessary for the enzyme to aminoacylate its tRNA. The codevelopment of a synthetase with its cognate tRNAs is necessary for the preservation of the genetic code and is supported by studies of various tRNA-synthetase interactions (7)(8)(9)(10)(11)(12). Thus, the E. coli enzyme has developed to recognize tRNA determinants of E. coli tRNA Cys , while the yeast enzyme has developed to recognize determinants of yeast tRNA Cys .…”