Purification and Primary Structure Determination of the N‐Terminal Blocked Protein, L11, from <i>Escherichia coli</i> Ribosomes

Dognin, Mai J.; Wittmann‐Liebold, Brigitte

doi:10.1111/j.1432-1033.1980.tb04995.x

Cited by 50 publications

(25 citation statements)

References 69 publications

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“…Thus, the next step undertaken was to consider commonly observed post-translational modifications, especially those known or suspected to be highly conserved within this family of proteins. Among ribosomal proteins, post-translational modification of L3, L11, S12 and L7/ L12 are well-documented [19,18,[23][24][25][26][27]. The commonly observed post-translation modifications of ribosomal proteins in prokaryotic organisms are methylation, acetylation, and β-methylthiolation.…”

Section: Category 3: Common or Conserved Post-translational Modificatmentioning

confidence: 99%

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

et al. 2005

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A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.

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Section: Category 3: Common or Conserved Post-translational Modificatmentioning

confidence: 99%

“…In E. coli ribosomal protein L11 is trimethylated at three amino acid positions by a single enzyme, L11 methyltransferase [25]. Evaluating the experimental data from Fig.…”

Section: Category 3: Common or Conserved Post-translational Modificatmentioning

confidence: 99%

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

et al. 2005

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“…Here, the Escherichia coli ribosomal protein L11 contains an N-terminal trimethylalanine residue and trimethylated lysine residues at positions 3 and 39 (70). These modifications appear to be dependent upon the PrmA gene product of E. coli (71).…”

Section: Unusual Dual Protein Methyltransferases That May Recognize Bmentioning

confidence: 99%

The ribosome: A hot spot for the identification of new types of protein methyltransferases

Clarke¹

2018

Journal of Biological Chemistry

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Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for posttranslationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, while the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins. Regulation of biological function by protein methylation reactionsIt is more and more apparent just how much of biological function is dependent upon posttranslational modifications (1). The human genome encodes some 900 enzymes catalyzing just protein phosphorylation or ubiquitination (2,3). However, it is now clear that methyl groups can stand beside phosphate groups and ubiquitin as major players in controlling the physiological functions of proteins. We are beginning to understand how the much greater diversity of protein methylation reactions can give rise to a greater diversity of function (1,4-8). We are also learning the importance of crosstalk between protein and DNA methylation reactions (9) and among protein methylation, phosphorylation, acetylation, and ubiquitination reactions (10-12). Finally, we are discovering how alterations in protein methylation pathways can lead to human pathology, particularly in cancer (13-15).The collection of over sixty human protein arginine and lysine methyltransferases that leave histone "marks" recognized by reader proteins to guide gene expression are perhaps the poster children for the importance of protein methyltransferases (16-17). However, recent work has emphasized the importance of methylating non-histone substrates at these residues, particularly ribosomal proteins, translation factors, and transcription factors in a wide variety of organisms (7,8,15,18,19). Furthermore, the methylation of lysine and arginine residues represents just the tip of the iceberg in protein methylation -modifications have also been established at histidine, modified histidine (diphthamide), glutamate, glutamine, asparagine, Lisoaspartate, D-aspartate, cysteine, isoprenylcysteine, me...

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“…Escherichia coli L11 is trimethylated at three amino acid positions, specifically Ala1 (the N-terminal methionine is removed posttranslationally), Lys3, and Lys39, acquiring a total of nine methyl groups (11,17). These methyl groups are added by a single enzyme, the L11 methyltransferase PrmA, encoded by the prmA gene (7,27).…”

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Thermus thermophilus L11 Methyltransferase, PrmA, Is Dispensable for Growth and Preferentially Modifies Free Ribosomal Protein L11 Prior to Ribosome Assembly

et al. 2004

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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.Extensive modification is a feature of many components of the translational apparatus, with the most common being methylation. In addition to methylated ribosomal RNAs, several ribosomal proteins are also methylated or acetylated. The most highly methylated protein component of the bacterial translational apparatus is the 50S ribosomal subunit protein L11 (2), which comprises a major part of the factor-binding region of the 50S subunit. Cryo-electron microscopic reconstructions of ribosome-EF-Tu (22, 25) and ribosome-EF-G (1) complexes demonstrated a dynamic conformation of L11 and a direct contact of the L11 region of the ribosome with both elongation factors. How, or even whether, methylation of L11 influences its function in multiple aspects of protein synthesis is not yet understood.Escherichia coli L11 is trimethylated at three amino acid positions, specifically Ala1 (the N-terminal methionine is removed posttranslationally), Lys3, and Lys39, acquiring a total of nine methyl groups (11, 17). These methyl groups are added by a single enzyme, the L11 methyltransferase PrmA, encoded by the prmA gene (7,27). This protein is conserved among bacteria but is absent from eukarya and archaea (4). Nevertheless, a prmA null mutant of E. coli has no detectable phenotype (27), leaving open the question of the function of this modification.The substrate recognition mechanism of this enzyme is of particular interest because it methylates multiple positions on the same protein. Presumably, the sites of methylation exist in structurally similar contexts, although the N terminus of L11 in the X-ray crystal structure (29) is disordered, preventing a definitive answer to this question. It is ...

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Purification and Primary Structure Determination of the N‐Terminal Blocked Protein, L11, from Escherichia coli Ribosomes

Cited by 50 publications

References 69 publications

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

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

The ribosome: A hot spot for the identification of new types of protein methyltransferases

Thermus thermophilus L11 Methyltransferase, PrmA, Is Dispensable for Growth and Preferentially Modifies Free Ribosomal Protein L11 Prior to Ribosome Assembly

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