Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

Suh, Moo-Jin; Hamburg, Daisy-Malloy; Gregory, Steven T.; Dahĺberg, Albert E.; Limbach, Patrick A.

doi:10.1002/pmic.200402111

Cited by 54 publications

(72 citation statements)

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“…We examined the modification states of several S12 mutants of T. thermophilus IB-21 (ATCC 43815) (16) by MALDI-TOF MS as described previously (23). Wild-type S12 was determined to have a mass of 14,519 Ϯ 6 Da (Table 1), consistent with our previous report (24), indicating loss of the initial methionine and the presence of the ␤-methylthiolation. Considering the proximity to D88, we sought to determine if the streptomycinresistant mutants K87R and K87E (12) were modified at D88.…”

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confidence: 52%

“…2A), near the streptomycin binding site and in the midst of residues altered in streptomycin-resistant mutants (14). This modification has also been found to occur in the phototrophic bacterium Rhodopseudomonas palustris (22), and we have identified it for the extremely thermophilic bacterium T. thermophilus (24). Posttranscriptional modifications of rRNA residues have been shown to affect resistance to various antibiotic classes (reviewed in reference 8).…”

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confidence: 74%

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Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

et al. 2006

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Ribosomal protein S12 contains a highly conserved aspartic acid residue that is posttranslationally ␤-methylthiolated. Using mass spectrometry, we have determined the modification states of several S12 mutants of Thermus thermophilus and conclude that ␤-methylthiolation is not a determinant of the streptomycin phenotype.Streptomycin and streptomycin-resistant mutants have played a seminal role in the elucidation of the decoding process of protein synthesis (reviewed in reference 19). Genetic and biochemical analyses of ribosomes from streptomycin-resistant mutants implicated ribosomal protein S12 as the determinant of the various streptomycin phenotypes, including resistance, dependence, and pseudodependence (reviewed in references 11 and 17). Such mutations have been localized in the three-dimensional structure of the Thermus thermophilus 30S ribosomal subunit to reside within two highly conserved loops centered around residues P90 and K42 (Escherichia coli numbering used throughout) ( Fig. 1) (7).It has only recently been discovered by use of mass spectrometry that E. coli ribosomal protein S12 is posttranslationally modified via a ␤-methylthiolation at position D88 ( Fig. 2A), near the streptomycin binding site and in the midst of residues altered in streptomycin-resistant mutants (14). This modification has also been found to occur in the phototrophic bacterium Rhodopseudomonas palustris (22), and we have identified it for the extremely thermophilic bacterium T. thermophilus (24). Posttranscriptional modifications of rRNA residues have been shown to affect resistance to various antibiotic classes (reviewed in reference 8). For example, ksgA mutants are resistant to kasugamycin due to the loss of N6-dimethylation of two conserved adenosines in 16S rRNA (13), while methylation of rRNA in the peptidyltransferase center (21) or in the decoding region (3) confers resistance to erythromycin and aminoglycosides, respectively (reviewed in references 20 and 26).Interestingly, loss of modification of a ribosomal protein has not yet been shown to affect antibiotic sensitivity. Nevertheless, the proximity of S12 residue D88 to residues altered in streptomycin-resistant mutants raises the possibility that such mutations might confer resistance indirectly by inhibiting ␤-methylthiolation of D88. To address this question, we performed matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to establish the modification status of ribosomal protein S12 in a series of streptomycinresistant and streptomycin-dependent mutants of T. thermophilus.Loss of ␤-methylthiolation in a subset of S12 mutants. We examined the modification states of several S12 mutants of T. thermophilus IB-21 (ATCC 43815) (16) by MALDI-TOF MS as described previously (23). Wild-type S12 was determined to have a mass of 14,519 Ϯ 6 Da (Table 1), consistent with our previous report (24), indicating loss of the initial methionine and the presence of the ␤-methylthiolation. Considering the proximity to D88, we sought to deter...

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confidence: 74%

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

et al. 2006

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“…Intact proteins were assigned by accurate mass measurement of the ribosome mixture before proteolysis (T = 0 min) and by comparison to protein molecular weights predicted for T. thermophilus HB8 from entries in the SwissProt database as before [42]. The r-proteins L1, L2, L3, L6, L13, L14, L15, L16, L17, L18, L20, L21, L22, L23, L24, L28, L29, L30, L32, L33, L34, L35, and L36 of the large subunit, and rproteins SThx, S6, S8, S9, S10, S14, S15 and S17 of the small subunit were still detected as intact proteins even after incubation with either trypsin or Proteinase K for 1500 min.…”

Section: Limited Proteolysis Of T Thermophilus 70s Ribosomesmentioning

confidence: 99%

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Suh

Pourshahian

Limbach

2007

J. Am. Soc. Mass Spectrom.

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An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30-60 min of incubation provide insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.The ribosome, found in all organisms, is the subcellular organelle that performs the activity of protein synthesis. Prokaryotic ribosomes, which sediment at 70S, have a molecular mass of approximately 2.5 ×10 6 Da and consist of two subunits, the 50S and the 30S. Each subunit has a unique number of proteins and ribosomal RNAs (rRNAs). Eukaryotic ribosomes, which sediment at 80S, are substantially larger and more complex than their prokaryotic counterparts. Two subunits, the 60S and 40S, together contain at least 78 unique proteins and 4 rRNAs. The small subunit binds messenger RNA (mRNA) and mediates the interactions between mRNA and transfer RNAs (tRNAs). The larger subunit catalyzes peptide-bond formation. During the initiation phase of protein synthesis, the two subunits behave independently, assembling into complete ribosomes only when elongation is about to begin.A fundamental prerequisite for understanding the biochemical interactions occurring within the ribosome during protein synthesis is a detailed knowledge of the structure of this † Current address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10021 * To whom correspondence should be addressed. Phone (513) 556-1871, Fax (513) Email: Pat.Limbach@uc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

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“…Proteomic studies of bacterial ribosomal proteins have reported di-or monomethylation at this residue in E. coli (29), Caulobacter crescentus (28), and Rhodopseudomonas palustris (27), and the reported molecular mass of Thermus thermophilus RpL7/L12 also suggests methylation (26). However, this modification appears not to be present in all bacteria as Bacillus subtilis RpL7/L12 was found to be unmodified (45).…”

Section: Discussionmentioning

confidence: 99%

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

Małecki

Dahl

Moen

et al. 2016

Journal of Biological Chemistry

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Human METTL20 is a mitochondrial, lysine-specific methyltransferase that methylates the ␤-subunit of electron transfer flavoprotein (ETF␤). Interestingly, putative METTL20 orthologues are found in a subset of ␣-proteobacteria, including Agrobacterium tumefaciens. Using an activity-based approach, we identified in bacterial extracts two substrates of recombinant METTL20 from A. tumefaciens (AtMETTL20), namely ETF␤ and the ribosomal protein RpL7/L12. We show that AtMETTL20, analogous to the human enzyme, methylates ETF␤ on Lys-193 and Lys-196 both in vitro and in vivo. ETF plays a key role in mediating electron transfer from various dehydrogenases, and we found that its electron transferring ability was diminished by AtMETTL20-mediated methylation of ETF␤. Somewhat surprisingly, AtMETTL20 also catalyzed monomethylation of RpL7/L12 on Lys-86, a common modification also found in many bacteria that lack METTL20. Thus, we here identify AtMETTL20 as the first enzyme catalyzing RpL7/ L12 methylation. In summary, here we have identified and characterized a novel bacterial lysine-specific methyltransferase with unprecedented dual substrate specificity within the seven ␤-strand class of lysine-specific methyltransferases, as it targets two apparently unrelated substrates, ETF␤ and RpL7/L12. Moreover, the present work establishes METTL20-mediated methylation of ETF␤ as the first lysine methylation event occurring in both bacteria and humans.

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Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

Cited by 54 publications

References 35 publications

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

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