Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis - PubMed (original) (raw)

Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis

Sharon Y Wong et al. Antimicrob Agents Chemother. 2013 Dec.

Abstract

Posttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular-the methylation of guanosine at position 518 (G518) of the 16S rRNA in Mycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidB mutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidB mutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility of M. tuberculosis to streptomycin (SM). Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidB mutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidB mutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed in M. tuberculosis strains that contain a gidB mutation.

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Figures

Fig 1

(A) Crystal structure of the 30S ribosomal subunit of Thermus thermophilus complexed with mRNA and cognate tRNA in the A site (Protein Data Bank [PDB] accession no. 1IBM). 16S rRNA is shown in gray, the A site is in orange, the S12 ribosomal protein is in blue, tRNA is in dark green, and mRNA is in light green. The black box highlights the region shown in panel B. (B) The N7 atom (pink) of G527 (magenta balls and sticks), which corresponds to G518 in M. tuberculosis, is approximately 5 Å; from the C4 atom (turquoise) of proline 44 (blue balls and sticks) of the S12 ribosomal protein (blue tube). The S12 ribosomal protein interacts with the wobble position (highlighted in yellow) of the mRNA:tRNA codon:anticodon helix (dark and light green) (9).

Fig 2

A comparative LC-MS (multiple-stage mass spectrometry) analysis of the RNase T1 digest of 16S rRNA purified from strain H37Rv (A) and strain Δ_gidB_ (B) shown to scale. (A) The top chromatogram is the total ion chromatogram (TIC) of the RNase T1 digest of 5 μg of 16S rRNA from H37Rv. The middle chromatogram shows a well-defined peak for m/z 817.6, which corresponds to the modification of the specific oligonucleotide CC[mG]CG. The bottom chromatogram shows the peak for multiple-stage mass spectrometry of the analyte ion, where m/z 817.6 was subjected to a first stage of fragmentation (MS/MS) and the resulting most abundant fragment ion m/z 735.1 was subjected to a second stage of fragmentation (MS/MS/MS). Only an ion that generates m/z 735.1 from m/z 817.6 yields a response in this analysis. (B) The top chromatogram is the TIC of the RNase T1 digest of 7 μg of 16S rRNA from strain Δ_gidB_. The middle chromatogram does not show a well-defined peak for m/z 817.6. The bottom chromatogram corresponds to the multiple-stage mass spectrometry of the analyte ions as described above. The absence of any response represented in the bottom chromatogram indicates the lack of a methylguanine base loss from the oligonucleotides that yielded a response in the first stage of tandem mass spectrometry shown in the middle chromatogram.

Fig 3

(A) Electrospray mass spectrum corresponding to the peak seen in the extracted ion chromatogram for m/z 817.6 of the RNase T1 digest of 5 μg of 16S rRNA from H37Rv M. tuberculosis (Fig. 2A, middle chromatogram). The mass spectral data are consistent with the doubly charged ion that would be expected for CC[mG]CG. The other m/z values in this mass spectrum correspond to additional RNase T1 digestion products from 16S rRNA. (B) Collision-induced dissociation (CID) mass spectrum of the RNase T1 digestion product at m/z 817.6 shown in panel A. (C) CID mass spectrum of the fragment with m/z 735.1 shown in panel B. The observed sequence-informative fragments correspond to the expected fragmentation pattern of oligonucleotide CC[mG]CG, which has an mG-base loss. The absence of mG is depicted as [G] in the sequence representation. Sequence-informative fragment ions are labeled following the nomenclature of McLuckey et al. (31).

Fig 4

Binding isotherms obtained from isothermal titration calorimetry analysis of streptomycin (SM) bound to H37Rv wild-type (top plot) or Δ_gidB_ mutant (bottom plot) 70S ribosomes. SM binds to wild-type ribosomes with a binding affinity that is 2 orders greater than that seen with the binding of SM to mutant ribosomes. (strain H37Rv Kd = 0.8 nM, with a confidence interval of 4.3 nM or greater; strain Δ_gidB Kd_ = 340 nM, with a confidence interval of 147 nM to 787 nM).

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Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis - PubMed (original) (raw)