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

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Kaitlyn Tsai ◽  
Vanja Stojković ◽  
Lianet Noda-Garcia ◽  
Iris D Young ◽  
Alexander G Myasnikov ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Ribosomal Rna ◽  
Binding Sites ◽  
Molecular Basis ◽  
Direct Evidence ◽  
Resistance Mechanism ◽  
Cryo Electron Microscopy ◽  
New Antibiotics ◽  
Natural Isolates ◽  
Adenosine Nucleotide

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

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 Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariam Jaafar ◽  
Julia Contreras ◽  
Carine Dominique ◽  
Sara Martín-Villanueva ◽  
Régine Capeyrou ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Rna Helicase ◽  
Genetic Interactions ◽  
Large Ribosomal Subunit ◽  
Base Pairing ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center

AbstractSynthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

scholarly journals A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

2014 ◽  
Vol 207 (4) ◽  
pp. 481-498 ◽  
Author(s):  
Jochen Baßler ◽  
Helge Paternoga ◽  
Iris Holdermann ◽  
Matthias Thoms ◽  
Sander Granneman ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Ribosome Assembly ◽  
Functional Importance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Assembly Factors

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.

Histidine 229 in protein L2 is apparently essential for 50S peptidyl transferase activity

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1087-1094 ◽  
Author(s):  
Barry S. Cooperman ◽  
Tammy Wooten ◽  
Robert R. Traut ◽  
Daniel P. Romero
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Direct Evidence ◽  
Protein Composition ◽  
Complete Loss ◽  
The Other ◽  
Transferase Activity ◽  
Wild Type ◽  
Peptidyl Transferase ◽  
Site Specific

It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the work reported here, we reconstitute 50S subunits in which the H229Q variant of L2 replaces L2, with all other ribosomal components remaining unchanged, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserved His 229 to Gin results in a complete loss of peptidyl transferase activity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activity. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identical with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S subunits. Furthermore, as shown by chemical footprinting analysis, H229Q-L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, the effect of H229 mutation appears to be confined to an effect on peptidyl transferase activity, providing the most direct evidence for protein involvement in this function to date.Key words: protein L2, site-specific mutagenesis, peptidyl transferase, reconstitution, histidine.

scholarly journals Fungal β-Tubulin, Expressed as a Fusion Protein, Binds Benzimidazole and Phenylcarbamate Fungicides

1998 ◽  
Vol 42 (9) ◽  
pp. 2171-2173 ◽  
Author(s):  
Derek W. Hollomon ◽  
Jenny A. Butters ◽  
Helen Barker ◽  
Len Hall
Keyword(s):  
Fusion Protein ◽  
Binding Sites ◽  
Molecular Basis ◽  
Plant Disease ◽  
Gel Filtration ◽  
Single Amino Acid ◽  
Cross Resistance ◽  
Plant Disease Control ◽  
First Time ◽  
Β Tubulin

ABSTRACT Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in β-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing β-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to β-tubulin. This binding is reduced by the mutation Glu198→Gly198, which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.

scholarly journals The Ancient History of Peptidyl Transferase Center Formation as Told by Conservation and Information Analyses

Life ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 134 ◽  
Author(s):  
Francisco Prosdocimi ◽  
Gabriel S. Zamudio ◽  
Miryam Palacios-Pérez ◽  
Sávio Torres de Farias ◽  
Marco V. José
Keyword(s):  
Ribosomal Rna ◽  
3D Structure ◽  
Pairwise Alignment ◽  
23S Rrna ◽  
Peptidyl Transferase ◽  
Tertiary Structures ◽  
Peptidyl Transferase Center ◽  
Universal Common Ancestor ◽  
Complete Sequences ◽  
23S Ribosomal Rna

The peptidyl transferase center (PTC) is the catalytic center of the ribosome and forms part of the 23S ribosomal RNA. The PTC has been recognized as the earliest ribosomal part and its origins embodied the First Universal Common Ancestor (FUCA). The PTC is frequently assumed to be highly conserved along all living beings. In this work, we posed the following questions: (i) How many 100% conserved bases can be found in the PTC? (ii) Is it possible to identify clusters of informationally linked nucleotides along its sequence? (iii) Can we propose how the PTC was formed? (iv) How does sequence conservation reflect on the secondary and tertiary structures of the PTC? Aiming to answer these questions, all available complete sequences of 23S ribosomal RNA from Bacteria and Archaea deposited on GenBank database were downloaded. Using a sequence bait of 179 bp from the PTC of Thermus termophilus, we performed an optimum pairwise alignment to retrieve the PTC region from 1424 filtered 23S rRNA sequences. These PTC sequences were multiply aligned, and the conserved regions were assigned and observed along the primary, secondary, and tertiary structures. The PTC structure was observed to be more highly conserved close to the adenine located at the catalytical site. Clusters of interrelated, co-evolving nucleotides reinforce previous assumptions that the PTC was formed by the concatenation of proto-tRNAs and important residues responsible for its assembly were identified. The observed sequence variation does not seem to significantly affect the 3D structure of the PTC ribozyme.

scholarly journals Disruption of evolutionarily correlated tRNA elements impairs accurate decoding

2020 ◽  
Vol 117 (28) ◽  
pp. 16333-16338
Author(s):  
Ha An Nguyen ◽  
S. Sunita ◽  
Christine M. Dunham
Keyword(s):  
Ribosomal Rna ◽  
Molecular Basis ◽  
Anticodon Loop ◽  
Wild Type ◽  
Stem Loop ◽  
Transfer Rnas ◽  
Translational Accuracy ◽  
Bacterial Ribosome ◽  
Evolutionarily Conserved ◽  
23S Ribosomal Rna

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem−loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-typetRNAGGCAlaortRNAGGCAlacontaining a reversed 32–38 pair on cognate and near-cognate codons. Structures of wild-typetRNAGGCAlabound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon−anticodon interaction is cognate or near cognate. Further, the 32–38 pair is destabilized in the context of a near-cognate codon−anticodon pair. Reversal of the pairing intRNAGGCAlaablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32–38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

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