Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine.

Abstract

RNA hairpin helices whose sequences are based on the acceptor stems of alanine and histidine tRNAs are specifically aminoacylated with their cognate amino acids. In these examples, major determinants for the identities of the respective tRNAs reside in the acceptor stem; the anticodon and other parts of the tRNA are dispensable for aminoacylation. In contrast, the anticodon is a major determinant for the identity of a methionine tRNA. RNA hairpin helices and hybrid duplexes that reconstruct the acceptor-TlWC stem and the acceptor stem, respectively, of methionine tRNA were investigated here for aminoacylation with methionine. Direct visualization of the aminoacylated RNA product on an acidic polyacrylamide gel by phosphor imaging demonstrated specific aminoacylation with substrates that contained as few as 7 base pairs. No aminoacylation with methionine was detected with several analogous RNA substrates whose sequences were based on noncognate tRNAs. While the efficiency of aminoacylation is reduced by orders of magnitude relative to methionine tRNA, the results establish that specific aminoacylation with methionine of small duplex substrates can be achieved without the anticodon or other domains of the tRNA. The results, combined with earlier studies, suggest a highly specific adaptation of the structures of aminoacyl-tRNA synthetases to the acceptor stems of their cognate tRNAs, resulting in a relationship between the nucleotide sequences/structures of small RNA duplexes and specific amino acids.Although nucleotides scattered throughout the threedimensional structures of transfer RNAs have been implicated as important for recognition by aminoacyl-tRNA synthetase (1, 2), much emphasis has been placed on interactions with the acceptor stem (3-6) and, additionally or alternatively, the anticodon (7-16). These elements are located in the two distinct domains of the L-shaped tRNA structure. In the cocrystal of Escherichia coli glutamine-tRNA synthetase with tRNAGIn (15,17) and also of yeast aspartyl-tRNA synthetase complexed with tRNAASP (16) these tRNA structural elements are in contact with distinct domains of the protein. A similar segregation of acceptor helix-anticodon interactions is proposed to occur in the complex of E. coli methionine-tRNA synthetase with tRNAMet (18). Although there is no crystal structure for E. coli alanine-tRNA synthetase, RNA footprinting revealed that the anticodon does not contact the bound protein (19). In this system, an acceptor helix G3-U70 base pair is a major determinant for recognition (3). RNA minihelices and duplexes that reconstruct part or all of acceptor-TTC or acceptor helix are aminoacylated with alanine, provided they contain G3-U70 (5,20,21). Specifically, the exocylic 2-amino group of G3 in the acceptor helix projects into the minor groove and is essential for interaction with alanine-tRNA synthetase (22).Similarly, small hairpin helices that correspond to the acceptor stem of E. coli tRNAHis are aminoacylated with histidine (6). Aminoacylation requires an e...

Striking effects of coupling mutations in the acceptor stem on recognition of tRNAs by Escherichia coli Met-tRNA synthetase and Met-tRNA transformylase.

“…Alternatively, even for tRNAs containing the CAU anticodon, aminoacylation in vivo may be somewhat sensitive to changes in the acceptor stem. Martinis and Schimmel (26) have shown recently that minihelix and microhelix variants of acceptor stem of tRNAMet can be aminoacylated in vitro with methionine, although at an extremely slow rate, and that mutations in the acceptor stem sequence abolish this aminoacylation.…”

Covalent methionylation of escherichia coli methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry

“…It should be noted that supporting evidence for covalent reaction of methionine with methionyl-tRNA synthetase has been already briefly described by Martinis and Schimmel (1992).…”

A ribonucleopeptide world at the origin of life

“…Previous evidence showed that a T box riboswitch can bind an aminoacylated tRNA without inducing gene expression (Grundy et al, 2005), and that some T box riboswitches can specifically recognize up to two tRNA molecules (Gutierrez-Preciado et al, 2009;Saad et al, 2013). It is then possible that in a primitive RNPep world, where tRNA minihelices-with primitive stem loop structures, and with reduced structural hindrance in comparison to modern tRNA-are aminoacylated (Martinis & Schimmel, 1992;Root-Bernstein et al, 2016), the T box riboswitch could then fit two aminoacylated tRNA minihelices that under the correct positional orientation allows the formation of a peptide bond between the two attached amino acids. Previous RNA selection experiments isolated ribozymes capable of catalyzing amino acid/peptide-RNA (flexizymes: Goto & Suga, 2009) or amino acid-amino acid binding (peptidyl-transferase ribozyme: Zhang & Cech, 1998).…”