| 1. |
- Seely, Savannah M., et al.
(författare)
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Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.
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| 2. |
- Abdalaal, Hind, et al.
(författare)
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Collateral toxicity limits the evolution of bacterial Release Factor 2 towards total omnipotence
- 2020
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:10, s. 2918-2930
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Tidskriftsartikel (refereegranskat)abstract
- When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.
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| 3. |
- Pundir, Shreya, et al.
(författare)
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The compensatory mechanism of a naturally-evolved E167K RF2 counteracting the loss of RF1 in bacteria
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The mechanism by which the naturally evolved E167K RF2 decodes RF1-specific UAG and Tryptophan (UGG) codons remains an open question. Our fast-kinetics-based fluorescent-peptide release assay reveals that E167K RF2 reads near-cognate UAG and UGG codons with significantly higher efficiency (221-530 folds) than the wild-type (WT) RF2, accompanied by dramatic drops in KM (Michaelis-Menten constant). Additionally, E167K RF2 is about four times slower in ribosomal turnover than WT RF2, which also indicates its higher affinity for the ribosome. Our 2.8 Å cryo-EM structure of E167K RF2 bound to the UGG-termination complex identifies unique stabilizing interactions between K167 and rRNA along with a new conformation of the conserved R213 in the decoding center, together justifying how E167K RF2 recognizes guanine as the third base of the codon, unlike WT RF2. Furthermore, thermal melting and SEC-SAXS assays suggest a somewhat destabilized con- formation of unbound E167K RF2. In conclusion, E167K RF2 demonstrates 'omnipotence' in recognizing all three stop codons and concurrently exhibits 'collateral toxicity' owing to its notably enhanced ribosome binding affinity and destabilized compact conformation, that enables it to bypass the fidelity checkpoint on UAG and UGG codons.
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| 4. |
- Li, Wen, et al.
(författare)
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Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G
- 2015
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Ingår i: Science Advances. - : AMER ASSOC ADVANCEMENT SCIENCE. - 2375-2548. ; 1:4
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Tidskriftsartikel (refereegranskat)abstract
- During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.
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| 5. |
- Liljas, Anders, et al.
(författare)
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The enigmatic ribosomal stalk
- 2018
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Ingår i: Quarterly reviews of biophysics (Print). - : Cambridge University Press. - 0033-5835 .- 1469-8994. ; 51
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Forskningsöversikt (refereegranskat)abstract
- The large ribosomal subunit has a distinct feature, the stalk, extending outside the ribosome. In bacteria it is called the L12 stalk. The base of the stalk is protein uL10 to which two or three dimers of proteins bL12 bind. In archea and eukarya P1 and P2 proteins constitute the stalk. All these extending proteins, that have a high degree of flexibility due to a hinge between their N- and C-terminal parts, are essential for proper functionalization of some of the translation factors. The role of the stalk proteins has remained enigmatic for decades but is gradually approaching an understanding. In this review we summarise the knowhow about the structure and function of the ribosomal stalk till date starting from the early phase of ribosome research.
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| 6. |
- Parajuli, Narayan Prasad, 1989-, et al.
(författare)
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Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.
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| 7. |
- Parajuli, Narayan Prasad, 1989-
(författare)
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Molecular Interplay of Antibiotics on the Bacterial Ribosome
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Inhibition of protein synthesis is one of the most common modes of action for medically useful antibiotics. This thesis presents the mechanistic studies of two chemically distinct classes of antibiotics that target bacterial ribosomes –aminoglycosides and thermorubin. Arbekacin (ABK) and amikacin (AMK) are two new-generation semisynthetic aminoglycosides (AGAs) that were developed to overcome enzyme-mediated AGA resistance in bacteria. Our results demonstrate that these two antibiotics induce potent inhibitory effects on various phases of bacterial protein synthesis. The binding of ABK stalls elongating ribosomes to a state that is unfavorable for elongation factor-G (EF-G) binding, which drastically prolongs the time for translocation from ~50 milliseconds to at least 2 seconds. ABK also abolishes the accuracy of mRNA decoding and inhibits peptide release. The results of in vitro fast kinetics and structures of ABK and AMK-bound 70S ribosomes reveal that in addition to canonical binding at h44 of 16S rRNA, appended amino-hydroxy butyryl (AHB) moiety of ABK and AMK secures extra interactions at the binding pocket and provides long dwelling-time on the translating ribosome. Moreover, AMK binds at the large subunit of ribosome proximal to the 3'CCA-end of the tRNA in the P-site and inhibits the release factor-mediated peptide release. Our data suggest that AGA impose bacteriostatic effects mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors. We have further characterized the molecular mechanism of action of the antibiotic thermorubin (THB) using in vitro fast kinetics and cryo-EM. We found that THB impedes elongation, termination, and ribosome recycling phases of translation. THB does so by binding to the intersubunit bridge B2a and extruding C1914 of H69 of 23S rRNA that interferes with the interactions of A-site substrates including aminoacyl-tRNAs, class-I release factors, and ribosome recycling factor. We also found that THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. These studies altogether suggest that in-depth characterization of antibiotic action provides important clues that hopefully aid in the development of new antibiotics to fight against looming antibiotic resistance.
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| 8. |
- Pundir, Shreya
(författare)
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Evolutionary Mechanisms Shaping Bacterial Translation Termination
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Translation termination in bacteria involves precise reading of stop codons (UAA, UAG, UGA) and coordinated peptidyl-tRNA hydrolysis by the class-I release factors (RFs) on the ribosome (70S). This thesis investigates the evolutionary and post-translational modification mechanisms of these RFs and the concurrent effect on bacterial translation termination.Unlike eukaryotes with a single RF, bacteria have two RFs. Release factor 1 (RF1) reads UAA and UAG; release factor 2 (RF2) reads UAA and UGA codons. So, why do bacteria host two RFs? To answer this, we performed an in vivo evolution experiment to explore how RF2 evolution compensates for the loss of RF1. Characterization of the evolved RF2 mutants, specifically E167K RF2, using both in vivo and in vitro peptide release assay reveals its ability to read the RF1-specific UAG codon and also the tryptophan (UGG) codon, displaying a functional trade-off termed “collateral toxicity”. Further, fast-kinetics-based peptide release assay shows that E167K RF2 is generally efficient in peptide release on UAA and UGA but significantly more efficient on UAG and UGG than WT RF2. This increased efficiency is primarily due to the higher affinity of E167K RF2 to the 70S. Our 2.8 Å cryo-EM structures demonstrated K167 to be engaged in hydrogen bond interactions with the rRNA, that are absent in WT RF2 having E167. Further, the mutant displays somewhat destabilized conformation when unbound, bypassing the conformational change check-point and facilitating the reading of near-cognate UAG and UGG codons. Post-translational methylation on the conserved GGQ motif of RF1/2 increases the efficiency of translation termination, but its role in termination accuracy was unknown. We compared the methylated and unmethylated variants of RF1/2 for cognate and near-cognate codon recognition. The unmethylated RFs exhibited lower termination accuracy, likely caused by the loss of conformational stability in the absence of GGQ methylation.In summary, these studies reveal the compensatory evolution of E167K RF2 as a tighter binder of the ribosome with the destabilized compact conformation that enhances UAG reading at the expense of UGG reading. Additionally, our study shows that GGQ methylation maintains the conformational stability of RF2 and facilitates accurate stop codon recognition.
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| 9. |
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| 10. |
- Aguirre Rivera, Javier, 1989-, et al.
(författare)
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Real-time measurements of aminoglycoside effects on protein synthesis in live cells
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 118:9
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Tidskriftsartikel (refereegranskat)abstract
- The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.
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