Misacylation of pyrrolysine tRNA in vitro and in vivo

Abstract

Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.

Fig. 1

Cloverleaf representation of M. barkeri Fusaro tRNA^Pyl, Bos taurus mitochondrial tRNA^Ser, M. barkeri MS tRNA^Pyl and M. barkeri tRNA^Ala _UGC. Differences in M. barkeri MS tRNA^Pyl are indicated by arrows. The boxes indicate the unusual structure shared by the tRNAs.

Fig. 2

Misacylation of M. barkeri Fusaro tRNA^Pyl with serine in vitro. tRNA^Pyl and bacterial-type SerRS (●), tRNA^Pyl and methanogenic type SerRS (▲), no enzyme (◆).

Fig. 3

Incorporation of serine and Cyc in a β-galactosidase/luciferase fusion reporter protein. Bars represent UAG read-through efficiency: M. barkeri tRNA^Pyl and either M. barkeri bacterial like SerRS (B-SerRS), M. barkeri methanogenic SerRSs (M-SerRS), E. coli SerRS (Ec-SerRS) or an empty expression vector (pET15 empty). As positive control, M. barkeri tRNA^Pyl and M. barkeri PylRS (Mb-PylRS) in the presence of Cyc was measured. Negative controls include measurements with either M-SerRS or B-SerRS without PylT.

Fig. 4

Misacylation of M. barkeri MS tRNA^Pyl with alanine in vitro. (A) In vitro aminoacylation of wild type M. barkeri MS tRNA^Pyl. Total E. coli tRNA with E. coli AlaRS (■), M. barkeri MS tRNA^Pyl with E. coli AlaRS (▲), no enzyme (◆). (B) Minihelices based on the sequences of, from left to right, M. barkeri MS tRNA^Ala (tRNA^Ala), M. barkeri MS tRNA^Pyl (tRNA^Pyl), M. barkeri MS C4:G69 tRNA^Pyl mutant (tRNA^Pyl C4:G69). (C) In vitro aminoacylation of minihelices. MS tRNA^Ala minihelix with E. coli AlaRS (●), MS tRNA^Pyl minihelix with the E. coli AlaRS (▲), MS tRNA^Pyl C4:G69 mutant minihelix with E. coli AlaRS (◆).

Fig. 5

Suppression of an E. coli strain trpA94 with the M. barkeri MS tRNA^Pyl C4:G69 mutant. E. coli transformants were streaked on M9 medium (A) with Trp (B) without Trp. E. coli transformed with 1, C4:G69 M. barkeri MS tRNA^Pyl mutant; 2, M. barkeri MS tRNA^Pyl wild type; 3, empty plasmid; 4, tRNA^Ala amber suppressor.