PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine

Abstract

Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA(Pyl). Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA(Pyl) in EMSA with apparent K(d) values of 0.12 and 0.13 μM, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA(Pyl) binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA(Pyl) binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA(Pyl) binding. Further indicating the specificity of PylSn interaction with tRNA(Pyl), substitutions of conserved residues in tRNA(Pyl) in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA(Pyl).

FIGURE 1.

Representative microbes with sequenced genomes that contain the pyl genes. A and B, the archaeal genomes are deposited at NCBI under accession numbers NC_003552 (A) and NC_007955 (B). C and D, the bacterial genomes have accession numbers NC_011830 (C) and AASZ01001259 as well as AASZ01001367 (D). The proteobacterial example is a gut symbiont sequenced in a metagenomic study of a marine worm. In all examples but D, the pyl genes are found in an apparent single transcriptional unit. In D, the pylTScSn genes and the pylBCD genes are found on separate contigs (groups of overlapping clones). The number labeling each gene is the locus number in the annotated genome. The numbers below genes in A–C show the percentage of similarity for each gene product when aligned with the corresponding M. acetivorans pyl gene product, in D the percentage of similarity to the corresponding gene products in M. acetivorans is shown, followed by the percentage of similarity to D. hafniense pyl gene products. Genes involved in the genetic encoding of pyrrolysine are in red, and those in biosynthesis of pyrrolysine are in blue. The pylSn genes are shown in pink, which matches the shading of the homologous 5′ region of the archaeal pylS genes.

FIGURE 2.

Electrophoretic mobility of in vitro transcribed M. barkeri tRNA^Pyl incubated in the presence of M. barkeri PylS or Δ92PylS. Protein concentrations added to tRNA^Pyl are indicated at the bottom of each gel. A, electrophoretic mobility of 20 nm tRNA^Pyl following incubation with increasing concentrations of PylS. B, electrophoretic mobility of 0.5 μm tRNA^Pyl subsequent to incubation with PylS or Δ92PylS.

FIGURE 3.

Efficacy of D. hafniense PylSn or PylSc in forming electrophoretically detectable complexes with D. hafniense tRNA^Pyl. Protein concentrations employed are indicated at the bottom of each lane. A, electrophoretic mobility of 17 nm tRNA^Pyl after incubation with indicated amounts of PylSn. B, electrophoretic mobility of 0.5 μm tRNA^Pyl after incubation with PylSc or PylSn.

FIGURE 4.

Specificity of D. hafniense PylSn for binding tRNA^Pyl as measured by electrophoretic shift. The panels show electrophoretic mobility shift assay of 0 μm and 10 μm PylSn with 17 nm tRNA^Pyl (lanes 1 and 2); 0 μm and 10 μm PylSn with 30 nm B. taurus mitochondrial tRNA^Ser (lanes 3 and 4); and 0 μm and 10 μm PylSn with 30 nm tRNA^Leu (lanes 5 and 6). Both panels are from the same polyacrylamide gel.

FIGURE 5.

D. hafniense PylSn binds tRNA^Pyl even in the presence of competing RNA. The panels show electrophoretic mobility shift assay of 17 nm tRNA^Pyl incubated in the presence of 1 μm PylSn and the E. coli tRNA pool. The calculated concentration of E. coli tRNA added to each binding assay was 0, 0.25, 0.5, 0.85, or 1.7 μm (lanes 2–6). The tRNA pool was 30% of total RNA in the preparation.

FIGURE 6.

Base changes made in the D. hafniense tRNA^Pyl in the acceptor stem and anticodon stem and loop. Those bases known to directly contact PylSc residues (13) are shown with gray shading. Locations in which bases were changed are shown boxed in this figure. None of these bases proved essential for EMSA detectable binding by PylSn.

FIGURE 7.

Base changes made within the D. hafniense tRNA^Pyld-stem and T stem and loop. Targeted base positions are indicated by the boxed bases in the tRNA. Bases that directly interact with PylSc are shown with gray shading (13). The base changes that strongly affected PylSn binding to tRNA, but still showed detectable binding in EMSA, are underlined. Base changes that eliminated the tRNA ability to form an EMSA-detectable complex with PylSn are boxed.

FIGURE 8.

Representative EMSA gels showing the effects of several base changes in the D. hafniense tRNA^Pyl on PylSn binding. A, electrophoretic mobility of D. hafniense tRNA^Pyl incubated in the absence (odd-numbered lanes) or presence (even-numbered lanes) of 10 μm PylSn. The tRNA^Pyl variants carried substitutions in the anticodon stem and loop as indicated at the top of the gel for each pair of lanes. All lanes are from a single electrophoretic gel. B, the panels show electrophoretic mobility of tRNA^Pyl variants carrying substitutions in the variable loop when incubated in the presence of 10 μm PylSn (even-numbered lanes) or in its absence (odd-numbered lanes). C45CA*, lanes 9 and 10 contain tRNA^Pyl in which an additional A was inserted between C45 and A46.