Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly

Abstract

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.

FIG. 1.

Construction of T. thermophilus prmA null mutant. (A) Approximately 165 bp of the wild-type prmA gene (white) containing the SAM binding motif (light gray) was replaced by the heat-stable kanamycin resistance gene (htk; black). KpnI restriction sites are indicated by vertical lines, HindIII restriction sites are indicated by horizontal lines, and PstI restriction sites are indicated in gray. The resulting T. thermophilus strain was designated TLK90. (B) Diagnostic PCRs to confirm disruption of the prmA gene in strain TLK90. Genomic DNA from either wild-type HB8 (W), the null mutant (M), or plasmid pUC19prmA::htk (P) was used as a template for PCRs. The primer sets used for each reaction (A to G) are indicated and shown schematically along with the expected product sizes. The region of the prmA gene that was deleted from the null mutant is represented schematically in gray, and the htk gene is indicated in black. Amplification of the β-lactamase gene (β-lac) from the plasmid template but not from TLK90 genomic DNA template confirmed the absence of this plasmid-derived ampicillin resistance marker gene in the null mutant. Molecular weight DNA ladders are shown (L), and their sizes are indicated in base pairs.

FIG. 2.

MALDI-TOF MS of 70S ribosomes from T. thermophilus strain HB8 (top) and prmA::htk null mutant TLK90 (bottom). The portions of the spectra containing proteins in the mass range of 14,000 to 18,000 Da are shown. The peaks corresponding to L11 show masses of 15,675.1 and 15,506.2 Da for the wild type and the mutant, respectively. The mass difference of 169 Da is equivalent to the mass of 12 methyl groups.

FIG. 3.

(A) Purification of cloned His₆-tagged T. thermophilus L11 methyltransferase, PrmA, from E. coli BL21(DE3) carrying plasmid pET30bprmA. Lane 1, molecular size markers (sizes are indicated); lane 2, whole-cell lysate prior to induction; lane 3, whole-cell lysate after 2-h induction with IPTG; lane 4, cleared lysate after DNase treatment; lane 5, supernatant after 30-min incubation of lysate at 65°C; lane 6, purified PrmA after Ni-NTA purification, desalting, and concentration. (B). In vitro methylation of ribosomes from wild-type HB8 and the prmA null mutant, TLK90. Methylation assays were performed over a 90-min time course. ♦, ribosomes from wild-type HB8 (WT); ×, ribosomes from TLK90; +, reaction without ribosomes (minus 70S); −, ribosomes from TLK90 without the enzyme (minus PrmA). The results shown were obtained from at least three independent experiments. (C) ³H-labeled products after in vitro methylation of ribosomes from wild-type HB8 (WT) and the prmA null mutant (TLK90). Methylation assays were performed, with samples being removed after 30, 60, and 90 min and electrophoresed through a sodium dodecyl sulfate-15% polyacrylamide gel (1 pmol equivalent of ribosomes per lane). After fixing in a 10% methanol-acetic acid solution, the gels were incubated for 20 min in Enlightning rapid autoradiography enhancer (Perkin-Elmer), dried, and exposed to film for 72 h. The positions of molecular mass markers on the gel are indicated.

FIG. 4.

In vitro methylation of ribosomes from wild-type HB8 and the prmA null mutant (TLK90). The ribosomes were either intact, dissociated in a buffer containing a high salt concentration but lacking magnesium (buffer A), or dissociated by a treatment with RNase A. Methylation assays were performed for a 90-min time course. •, ribosomes from TLK90 in buffer A (TLK90 buf.A); ○, ribosomes from TLK90 treated with RNase A (TLK90 RNase); ×, ribosomes from TLK90; ▴, ribosomes from wild-type HB8 in buffer A (WT buf.A); ▪, ribosomes from wild-type HB8 treated with RNase A (WT RNase). Clustered near the bottom of the plot are ribosomes from wild-type HB8 (WT; filled diamonds), a reaction without ribosomes (+), and ribosomes from TLK90 without the enzyme (−). The results shown were obtained from at least three independent experiments.

FIG. 5.

Three-dimensional structure of Thermotoga maritima ribosomal protein L11 (blue) and its associated binding site on 23S rRNA (cyan) as determined by X-ray crystallography (29). Amino acid residues Lys16 and Lys39 are shown with red space-filling diagrams to indicate their mutual proximity. The N-terminal seven amino acid residues of L11, including Ala1 and Lys3, are disordered in the structure and are not shown.