scholarly journals Base-Pairing between 23S rRNA and tRNA in the Ribosomal A Site

1999 ◽  
Vol 4 (5) ◽  
pp. 859-864 ◽  
Author(s):  
Daniel F Kim ◽  
Rachel Green
Keyword(s):  
23S Rrna ◽  
Base Pairing ◽  
A Site

scholarly journals Oxazolidinone Antibiotics Target the P Site on Escherichiacoli Ribosomes

2002 ◽  
Vol 46 (4) ◽  
pp. 1080-1085 ◽  
Author(s):  
Hiroyuki Aoki ◽  
Lizhu Ke ◽  
Susan M. Poppe ◽  
Toni J. Poel ◽  
Elizabeth A. Weaver ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Wide Spectrum ◽  
Anaerobic Bacteria ◽  
Elongation Factor ◽  
Peptide Bond ◽  
23S Rrna ◽  
Peptide Bonds ◽  
A Site ◽  
Domain V ◽  
Trna Binding

ABSTRACT The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

scholarly journals Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 505-520 ◽  
Author(s):  
E. S. P. Bromfield ◽  
J. T. Tambong ◽  
S. Cloutier ◽  
D. Prévost ◽  
G. Laguerre ◽  
...  
Keyword(s):  
23S Rrna ◽  
Resistant Bacteria ◽  
Sweet Clover ◽  
Mineral Salts ◽  
Medicago Lupulina ◽  
Protein Encoding ◽  
History Of ◽  
Macroptilium Atropurpureum ◽  
A Site ◽  
Multiple Antibiotics

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency ∼68 %) but was recovered infrequently (∼1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species ‘Sinorhizobium morelense’. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

scholarly journals The expression of antibiotic resistance methyltransferase correlates with mRNA stability independently of ribosome stalling

2016 ◽  
pp. AAC.01806-16 ◽  
Author(s):  
Ekaterina Dzyubak ◽  
Mee-Ngan F. Yap
Keyword(s):  
Leader Peptide ◽  
23S Rrna ◽  
Cross Resistance ◽  
Nonsense Mutations ◽  
Stem Loop ◽  
Translational Initiation ◽  
Ribosome Stalling ◽  
Mrna Structure ◽  
Bacterial Ribosome ◽  
A Site

Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression ofermis activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the co-transcribederm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes theermShine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here we show that nonsense mutations in theStaphylococcus aureusErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence and presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of theermBL-ermBtranscript, and macrolide appears to mitigate mRNA cleavage at a site immediately downstream of theermBLSD sequence. The stabilizing effect of antibiotics on mRNA is not limited toermBL-ermB; cationic antibiotics representing a ribosome stalling inducer and a non-inducer increase the half-life of specific transcripts. These data unveil a new layer ofermBregulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

scholarly journals Interaction of Avilamycin with Ribosomes and Resistance Caused by Mutations in 23S rRNA

2002 ◽  
Vol 46 (11) ◽  
pp. 3339-3342 ◽  
Author(s):  
Christine B. Kofoed ◽  
Birte Vester
Keyword(s):  
Binding Site ◽  
Initiation Factor ◽  
23S Rrna ◽  
Cross Resistance ◽  
Halobacterium Halobium ◽  
Growth Promoter ◽  
A Site ◽  
Domain V ◽  
Trna Binding ◽  
Selection Of

ABSTRACT The antibiotic growth promoter avilamycin inhibits protein synthesis by binding to bacterial ribosomes. Here the binding site is further characterized on Escherichia coli ribosomes. The drug interacts with domain V of 23S rRNA, giving a chemical footprint at nucleotides A2482 and A2534. Selection of avilamycin-resistant Halobacterium halobium cells revealed mutations in helix 89 of 23S rRNA. Furthermore, mutations in helices 89 and 91, which have previously been shown to confer resistance to evernimicin, give cross-resistance to avilamycin. These data place the binding site of avilamycin on 23S rRNA close to the elbow of A-site tRNA. It is inferred that avilamycin interacts with the ribosomes at the ribosomal A-site interfering with initiation factor IF2 and tRNA binding in a manner similar to evernimicin.

scholarly journals Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site

2013 ◽  
Vol 110 (21) ◽  
pp. 8501-8506 ◽  
Author(s):  
J. M. Schifano ◽  
R. Edifor ◽  
J. D. Sharp ◽  
M. Ouyang ◽  
A. Konkimalla ◽  
...  
Keyword(s):  
23S Rrna ◽  
A Site

scholarly journals Phylogenetic Sequence Variations in Bacterial rRNA Affect Species-Specific Susceptibility to Drugs Targeting Protein Synthesis

2011 ◽  
Vol 55 (9) ◽  
pp. 4096-4102 ◽  
Author(s):  
Subramanian Akshay ◽  
Mihai Bertea ◽  
Sven N. Hobbie ◽  
Björn Oettinghaus ◽  
Dimitri Shcherbakov ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Drug Susceptibility ◽  
Drug Binding ◽  
23S Rrna ◽  
Content Type ◽  
Sequence Variations ◽  
Binding Pockets ◽  
Species Specific ◽  
Peptidyltransferase Center ◽  
A Site

ABSTRACTAntibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences inMycobacterium smegmatisribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket.

scholarly journals Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

2016 ◽  
Vol 113 (27) ◽  
pp. 7527-7532 ◽  
Author(s):  
Stefan Arenz ◽  
Manuel F. Juette ◽  
Michael Graf ◽  
Fabian Nguyen ◽  
Paul Huter ◽  
...  
Keyword(s):  
Single Molecule ◽  
Antimicrobial Agents ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Cross Resistance ◽  
Single Molecule Fret ◽  
Arginine Residues ◽  
Peptidyltransferase Center ◽  
A Site

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

scholarly journals Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

2009 ◽  
Vol 53 (4) ◽  
pp. 1411-1419 ◽  
Author(s):  
Ekaterini C. Kouvela ◽  
Dimitrios L. Kalpaxis ◽  
Daniel N. Wilson ◽  
George P. Dinos
Keyword(s):  
Antimicrobial Activity ◽  
Attachment Site ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Distinct Mode ◽  
Alkyl Aryl ◽  
Resistant Strains ◽  
Exit Tunnel ◽  
A Site

ABSTRACT Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin-resistant strains, while maintaining their activity against erythromycin-susceptible ones. In this study, we present a new ketolide, K-1325, that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies, K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild-type Escherichia coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the alkyl-aryl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increased affinity of U2609C mutant ribosomes for the drug and rationalizing the enhanced effectiveness of this new ketolide.

scholarly journals Insights into the base-pairing preferences of 8-oxoguanosine on the ribosome

2019 ◽  
Vol 47 (18) ◽  
pp. 9857-9870 ◽  
Author(s):  
Erica N Thomas ◽  
Carrie L Simms ◽  
Hannah E Keedy ◽  
Hani S Zaher
Keyword(s):  
Base Pair ◽  
Structural Changes ◽  
Bond Formation ◽  
Peptide Bond ◽  
Underlying Mechanism ◽  
Peptide Bond Formation ◽  
Base Pairing ◽  
Base Pairs ◽  
Free Dna ◽  
A Site

Abstract Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.

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