Aminoacylation of tRNA 2'- or 3'-hydroxyl by phosphoseryl- and pyrrolysyl-tRNA synthetases

Abstract

Class I and II aminoacyl-tRNA synthetases (AARSs) attach amino acids to the 2'- and 3'-OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl-tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2'-OH. Here we show that two Class II AARSs, O-phosphoseryl- (SepRS) and pyrrolysyl-tRNA (PylRS) synthetases, aminoacylate the 2'- and 3'-OH, respectively. Structure-based-phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2'-OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.

Keywords: Amino acid; Protein synthesis; tRNA esterification.

Figure 1. Site of amino acid attachment in M. maripaludis SepRS

Time course of aminoacylation of different tRNA^Cys species with Sep using M. maripaludis SepRS is shown. The tRNAs were modified at the 3′ termini as follows: tRNA lacking the 3′-terminal adenosine (∇), terminus reconstructed with 3′-deoxyadenosine (∎), 2′-deoxyadenosine (◊), and adenosine (•).

Figure 2. Analysis of the 3′ terminal adenosine modified M. barkeri Fusaro tRNA^Pyl transcripts charged with pyrrolysine (Pyl) in the presence (+) and absence ( ) M. barkeri Fusaro PylRS enzyme

The resulting tRNA products were loaded onto an acid gel and the samples were later exposed to a phosphoimager plate. The positions of tRNA^Pyl and Pyl-tRNA^Pyl are indicated. The lower band is the unmodified tRNA transcripts with a 3′-CC₇₅ end. 1 and 3 indicates the position of Pyl-tRNA^Pyl, while 2 and 4 indicates uncharged tRNA^Pyl. Mb, Methanosarcina barkeri; Dh, Desulfitobacterium hafniense.

Figure 3. The structure-based phylogenetic tree (calculated as in [28]) shows the evolutionary relationships between the class II AARS family and subclasses

The table of each aminoacyl-tRNA synthetase with its preference for the attachment of the amino acid to the 2′-OH or 3′-OH as compiled earlier [4] is combined with the experimentally determined specificity of PylRS and SepRS. Every member of the family directs aminoacylation to the 3′-OH of its cognate tRNA. PheRS, AsnRS, and SepRS are the only exceptions and preferentially aminoacylate the 2′-OH. The earliest point in evolution when the switch to 2′OH preference occurred is indicated (red arrow).

Figure 4. Structural modeling of PheRS, SepRS, and PylRS aminoacylation active sites

(A, B) A. fulgidus SepRS structure complexed with Sep (cyan, PDB: 2DU3) is superimposed onto the structure of T. thermophilus PheRS:tRNA^Phe:Phe-AMP analog complex (green, PDB: 2IY5). (C) The structure of Methanosarcina mazei PylRS complexed with Pyl (cyan, PDB: 2ZCE) is superimposed onto the structure of D. hafniense PylRS:tRNA^Pyl complex (green, PDB: 2ZNI).