scholarly journals Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3

2003 ◽  
Vol 47 (9) ◽  
pp. 2892-2896 ◽  
Author(s):  
Jacob Bøsling ◽  
Susan M. Poulsen ◽  
Birte Vester ◽  
Katherine S. Long
Keyword(s):  
Ribosomal Protein ◽  
Drug Binding ◽  
Mechanisms Of Resistance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Resistance Determinants ◽  
Drug Binding Site ◽  
Gene Coding ◽  
Bacterial Ribosome ◽  
Ribosomal Protein L3

ABSTRACT The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

scholarly journals Mutations in Ribosomal Protein L3 Are Associated with Oxazolidinone Resistance in Staphylococci of Clinical Origin

2009 ◽  
Vol 53 (12) ◽  
pp. 5275-5278 ◽  
Author(s):  
Jeffrey B. Locke ◽  
Mark Hilgers ◽  
Karen Joy Shaw
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Ribosomal Protein ◽  
Binding Site ◽  
Staphylococcus Epidermidis ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Ribosomal Protein L3

ABSTRACT Following recent reports of ribosomal protein L3 mutations in laboratory-derived linezolid-resistant (LZDr) Staphylococcus aureus, we investigated whether similar mutations were present in LZDr staphylococci of clinical origin. Sequence analysis of a variety of LZDr isolates revealed two L3 mutations, ΔSer145 (S. aureus NRS127) and Ala157Arg (Staphylococcus epidermidis 1653059), both occurring proximal to the oxazolidinone binding site in the peptidyl transferase center. The oxazolidinone torezolid maintained a ≥8-fold potency advantage over linezolid for both strains.

scholarly journals The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Purnima Klingauf-Nerurkar ◽  
Ludovic C Gillet ◽  
Daniela Portugal-Calisto ◽  
Michaela Oborská-Oplová ◽  
Martin Jäger ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Atpase Activity ◽  
Ribosomal Protein ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Ribosomal Stalk ◽  
Exit Tunnel ◽  
Ribosome Formation

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

scholarly journals Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

2021 ◽  
Author(s):  
Kaitlyn Tsai ◽  
Vanja Stojkovic ◽  
Lianet Noda-Garcia ◽  
Iris D. Young ◽  
Alexander G. Myasnikov ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Binding Sites ◽  
Molecular Basis ◽  
Direct Evidence ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Bacterial Ribosome ◽  
Mechanisms Of Adaptation

Many clinically useful antibiotics inhibit the bacterial ribosome. The ribosomal RNA-modifying enzyme Cfr methylates an adenosine (m8A2503) in the peptidyl transferase center and causes cross-resistance to several classes of antibiotics. Despite the prevalence of this mode of resistance, mechanisms of adaptation to antibiotic pressure that exploit ribosome modification by Cfr are poorly understood. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. To address these questions, we evolved Cfr under antibiotic selection to generate variants that confer increased resistance and methylation of rRNA, provided by enhanced Cfr expression and stability. Using a variant which achieves near-stoichiometric methylation, we determined a 2.2Å cryo-EM structure of the Cfr-modified ribosome, revealing the molecular basis for resistance and informing design of antibiotics that overcome Cfr resistance.

scholarly journals Mutations in 23S rRNA at the Peptidyl Transferase Center and Their Relationship to Linezolid Binding and Cross-Resistance

2010 ◽  
Vol 54 (11) ◽  
pp. 4705-4713 ◽  
Author(s):  
Katherine S. Long ◽  
Christian Munck ◽  
Theis M. B. Andersen ◽  
Maria A. Schaub ◽  
Sven N. Hobbie ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Synergistic Effects ◽  
23S Rrna ◽  
Cross Resistance ◽  
Resistance Pattern ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Linezolid Resistance ◽  
Bacterial Ribosome ◽  
Base Identity

ABSTRACT The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. The largest decrease in linezolid susceptibility for any of the introduced single mutations was observed with the G2576U mutation at a position that is 7.9 Å from linezolid. Smaller decreases were observed with the A2503G, U2504G, and G2505A mutations at nucleotides proximal to linezolid, showing that the degree of resistance conferred is not simply inversely proportional to the nucleotide-drug distance. The double mutations G2032A-C2499A, G2032A-U2504G, C2055A-U2504G, and C2055A-A2572U had remarkable synergistic effects on linezolid resistance relative to the effects of the corresponding single mutations. This study emphasizes that effects of rRNA mutations at the PTC are organism dependent. Moreover, the data show a nonpredictable cross-resistance pattern between linezolid, chloramphenicol, clindamycin, and valnemulin. The data underscore the significance of mutations at distal nucleotides, either alone or in combination with other mutated nucleotides, in contributing to linezolid resistance.

scholarly journals NAC covers ribosome-associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidyl transferase center.

1995 ◽  
Vol 130 (3) ◽  
pp. 519-528 ◽  
Author(s):  
S Wang ◽  
H Sakai ◽  
M Wiedmann
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Peptidyl Transferase ◽  
Nascent Polypeptide ◽  
Peptidyl Transferase Center ◽  
Protease Resistance ◽  
Protective Environment ◽  
Cytosolic Factors

We demonstrate that nascent polypeptide-associated complex (NAC) is one of the first cytosolic factors that newly synthesized nascent chains encounter. When NAC is present, nascent chains are segregated from the cytosol until approximately 30 amino acids in length, a finding consistent with the well-documented protease resistance of short ribosome-associated nascent chains. When NAC is removed, the normally protected nascent chains are susceptible to proteolysis. Therefore NAC, by covering COOH-terminal segments of nascent chains on the ribosome, perhaps together with ribosomal proteins, forms a protective environment for regions of nascent chains just emerging from the peptidyl transferase center. Since NAC is not a core ribosomal protein, the emergence of nascent chains from the ribosome may be more dynamic than previously thought.

scholarly journals The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics

2006 ◽  
Vol 50 (7) ◽  
pp. 2500-2505 ◽  
Author(s):  
Katherine S. Long ◽  
Jacob Poehlsgaard ◽  
Corinna Kehrenberg ◽  
Stefan Schwarz ◽  
Birte Vester
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Drug Binding ◽  
Animal Origin ◽  
23S Rrna ◽  
Peptidyl Transferase ◽  
E Coli ◽  
Peptidyl Transferase Center ◽  
Drug Classes

ABSTRACT A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.

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