Publications by authors named "Suparna Sanyal"

67 Publications

Mechanistic insights into translation inhibition by aminoglycoside antibiotic arbekacin.

Nucleic Acids Res 2021 Jun 14. Epub 2021 Jun 14.

Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, SE-75124 Uppsala, Sweden.

How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.
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http://dx.doi.org/10.1093/nar/gkab495DOI Listing
June 2021

Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis.

iScience 2021 May 20;24(5):102429. Epub 2021 Apr 20.

Institute of Molecular, Cell and Systems Biology, University of Glasgow College of Medical, Veterinary and Life Sciences, Glasgow G12 8QQ, UK.

The emergence of lipid membranes and embedded proteins was essential for the evolution of cells. Translocon complexes mediate cotranslational recruitment and membrane insertion of nascent proteins, but they already contain membrane-integral proteins. Therefore, a simpler mechanism must exist, enabling spontaneous membrane integration while preventing aggregation of unchaperoned protein in the aqueous phase. Here, we used giant unilamellar vesicles encapsulating minimal translation components to systematically interrogate the requirements for insertion of the model protein proteorhodopsin (PR) - a structurally ubiquitous membrane protein. We show that the N-terminal hydrophobic domain of PR is both necessary and sufficient for cotranslational recruitment of ribosomes to the membrane and subsequent membrane insertion of PR. Insertion of N-terminally truncated PR was restored by artificially attaching ribosomes to the membrane. Our findings offer a self-sufficient protein-inherent mechanism as a possible explanation for effective membrane protein biogenesis in a "pretranslocon" era, and they offer new opportunities for generating artificial cells.
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http://dx.doi.org/10.1016/j.isci.2021.102429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102411PMC
May 2021

Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation.

RNA Biol 2021 May 3:1-13. Epub 2021 May 3.

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA, with the natural dipeptidyl fMet-Phe-tRNA in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.
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http://dx.doi.org/10.1080/15476286.2021.1913312DOI Listing
May 2021

GGQ methylation enhances both speed and accuracy of stop codon recognition by bacterial class-I release factors.

J Biol Chem 2021 Apr 19:100681. Epub 2021 Apr 19.

Department of Cell and Molecular Biology, Uppsala University, Box-596, Biomedical Center, SE-75124, Uppsala, Sweden. Electronic address:

Accurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a 'compact to open' conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is post-translationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the 'compact to open' transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo.
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http://dx.doi.org/10.1016/j.jbc.2021.100681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131318PMC
April 2021

Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from 'Generalist' Ancestors.

Mol Biol Evol 2021 Apr 19. Epub 2021 Apr 19.

Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, SE-75124 Uppsala, Sweden.

It has been hypothesized that early enzymes are more promiscuous than their extant orthologues. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using Elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing pre-steady state fast kinetics using quench-flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3 to 3.3-billion-year-old ancestral EF-Tus towards two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. While the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. Also, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remain virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, 'generalist' ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.
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http://dx.doi.org/10.1093/molbev/msab114DOI Listing
April 2021

Real-time measurements of aminoglycoside effects on protein synthesis in live cells.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Department of Cell and Molecular Biology, Uppsala University, 752 36 Uppsala, Sweden

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 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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http://dx.doi.org/10.1073/pnas.2013315118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936356PMC
March 2021

Ribosomal RNA Modulates Aggregation of the Prion Protein HET-s.

Int J Mol Sci 2020 Sep 1;21(17). Epub 2020 Sep 1.

Department of Cell and Molecular Biology, Uppsala University, Box-596, Biomedical Center, 751 24 Uppsala, Sweden.

The role of the nucleic acids in prion aggregation/disaggregation is becoming more and more evident. Here, using HET-s prion from fungi () as a model system, we studied the role of RNA, particularly of different domains of the ribosomal RNA (rRNA), in its aggregation process. Our results using Rayleigh light scattering, Thioflavin T (ThT) binding, transmission electron microscopy (TEM) and cross-seeding assay show that rRNA, in particular the domain V of the major rRNA from the large subunit of the ribosome, substantially prevents insoluble amyloid and amorphous aggregation of the HET-s prion in a concentration-dependent manner. Instead, it facilitates the formation of the soluble oligomeric "seeds", which are capable of promoting de novo HET-s aggregation. The sites of interactions of the HET-s prion protein on domain V rRNA were identified by primer extension analysis followed by UV-crosslinking, which overlap with the sites previously identified for the protein-folding activity of the ribosome (PFAR). This study clarifies a missing link between the rRNA-based PFAR and the mode of propagation of the fungal prions.
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http://dx.doi.org/10.3390/ijms21176340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504336PMC
September 2020

Selective translation by alternative bacterial ribosomes.

Proc Natl Acad Sci U S A 2020 08 28;117(32):19487-19496. Epub 2020 Jul 28.

Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, 100084 Beijing, China;

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from , we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.
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http://dx.doi.org/10.1073/pnas.2009607117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431078PMC
August 2020

Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation.

Proc Natl Acad Sci U S A 2020 07 22;117(27):15609-15619. Epub 2020 Jun 22.

State Key Laboratory of Membrane Biology, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, 100871 Beijing, China;

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Δ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from Δ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the Δ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the Δ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.
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http://dx.doi.org/10.1073/pnas.1914323117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355043PMC
July 2020

Collateral Toxicity Limits the Evolution of Bacterial Release Factor 2 toward Total Omnipotence.

Mol Biol Evol 2020 10;37(10):2918-2930

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

When new genes evolve through modification of existing genes, there are often tradeoffs 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 highlights a functional tradeoff that we term "collateral toxicity." We suggest that this type of tradeoff may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary tradeoffs 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 to alterations in the demand not only for RF2 activity but also for RF1 activity.
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http://dx.doi.org/10.1093/molbev/msaa129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530605PMC
October 2020

Author Correction: Inhibition of translation termination by small molecules targeting ribosomal release factors.

Sci Rep 2020 Mar 13;10(1):4950. Epub 2020 Mar 13.

Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, SE-75124, Uppsala, Sweden.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-62010-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069971PMC
March 2020

Inhibition of translation termination by small molecules targeting ribosomal release factors.

Sci Rep 2019 10 28;9(1):15424. Epub 2019 Oct 28.

Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, SE-75124, Uppsala, Sweden.

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.
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http://dx.doi.org/10.1038/s41598-019-51977-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817905PMC
October 2019

RNA modulates aggregation of the recombinant mammalian prion protein by direct interaction.

Sci Rep 2019 08 27;9(1):12406. Epub 2019 Aug 27.

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Box-596, 75124, Sweden.

Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general.
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http://dx.doi.org/10.1038/s41598-019-48883-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712051PMC
August 2019

The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation.

Elife 2019 06 7;8. Epub 2019 Jun 7.

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

Applying pre-steady state kinetics to an based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.
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http://dx.doi.org/10.7554/eLife.46124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594751PMC
June 2019

Modulation of p53 and prion protein aggregation by RNA.

Biochim Biophys Acta Proteins Proteom 2019 10 28;1867(10):933-940. Epub 2019 Feb 28.

Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de Ciência Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil. Electronic address:

Several RNA-binding proteins undergo reversible liquid-liquid phase transitions, which, in pathological conditions, might evolve into transitions to solid-state phases, giving rise to amyloid structures. Amyloidogenic and prion-like proteins, such as the tumor suppressor protein p53 and the mammalian prion protein (PrP), bind RNAs specifically or nonspecifically, resulting in changes in their propensity to undergo aggregation. Mutant p53 aggregation seems to play a crucial role in cancer through loss of function, negative dominance and gain of function. PrP conversion modulated by RNA results in highly toxic aggregates. Here, we review data on the modulatory action of RNAs on the aggregation of both proteins.
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http://dx.doi.org/10.1016/j.bbapap.2019.02.006DOI Listing
October 2019

p53 Isoforms and Their Implications in Cancer.

Cancers (Basel) 2018 Aug 25;10(9). Epub 2018 Aug 25.

Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, Uppsala SE-75124, Sweden.

In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are ∆40p53, ∆133p53, and ∆160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered 'responsive elements' than p53. Furthermore, they may exert a 'dominant negative effect' or induce more aggressive cancer by the 'gain of function'. One possible mechanism of p53 inactivation can be through tetramerization with the ∆133p53 and ∆160p53 isoforms-both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation-possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.
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http://dx.doi.org/10.3390/cancers10090288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162399PMC
August 2018

Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A.

Nucleic Acids Res 2018 06;46(11):5861-5874

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of the monitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.
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http://dx.doi.org/10.1093/nar/gky346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009598PMC
June 2018

Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association.

Proc Natl Acad Sci U S A 2018 05 23;115(18):4649-4654. Epub 2018 Apr 23.

Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden

The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12-IF2 interaction on ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12-IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners-all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.
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http://dx.doi.org/10.1073/pnas.1802001115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939111PMC
May 2018

The enigmatic ribosomal stalk.

Q Rev Biophys 2018 01;51:e12

Department of Cell and Molecular Biology,Biomedical Center,Uppsala University,Uppsala,Sweden.

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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http://dx.doi.org/10.1017/S0033583518000100DOI Listing
January 2018

New melanocortin-like peptide of can suppress inflammation via the mammalian melanocortin-1 receptor (MC1R): possible endocrine-like function for microbes of the gut.

NPJ Biofilms Microbiomes 2017 13;3:31. Epub 2017 Nov 13.

Laboratory of Diabetes and Diabetes Related Research, US, USA.

releases a 33 amino acid peptide melanocortin-like peptide of (MECO-1) that is identical to the C-terminus of the elongation factor-G (EF-G) and has interesting similarities to two prominent mammalian melanocortin hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH). Note that MECO-1 lacks HFRW, the common pharmacophore of the known mammalian melanocortin peptides. MECO-1 and the two hormones were equally effective in severely blunting release of cytokines (HMGB1 and TNF) from macrophage-like cells in response to (i) endotoxin (lipopolysaccharide) or (ii) pro-inflammatory cytokine HMGB-1. The in vitro anti-inflammatoty effects of MECO-1 and of alpha-MSH were abrogated by (i) antibody against melanocortin-1 receptor (MC1R) and by (ii) agouti, an endogenous inverse agonist of MC1R. In vivo MECO-1 was even more potent than alpha-MSH in rescuing mice from death due to (i) lethal doses of LPS endotoxin or (ii) cecal ligation and puncture, models of sterile and infectious sepsis, respectively.
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http://dx.doi.org/10.1038/s41522-017-0039-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684143PMC
November 2017

Cryo-EM structure of Mycobacterium smegmatis ribosome reveals two unidentified ribosomal proteins close to the functional centers.

Protein Cell 2018 04;9(4):384-388

State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Center for Life Sciences, School of Life Sciences, Peking University, Beijing, 100871, China.

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http://dx.doi.org/10.1007/s13238-017-0456-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876184PMC
April 2018

R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria .

J Biol Chem 2017 09 25;292(36):15134-15142. Epub 2017 Jul 25.

From the Department of Cell and Molecular Biology, Uppsala University, Box-596, 75124 Uppsala, Sweden

The current understanding of the specificity of the bacterial class I release factors (RFs) in decoding stop codons has evolved beyond a simple tripeptide anticodon model. A recent molecular dynamics study for deciphering the principles for specific stop codon recognition by RFs identified Arg-213 as a crucial residue on RF2 for discriminating guanine in the third position (G3). Interestingly, Arg-213 is highly conserved in RF2 and substituted by Ile-196 in the corresponding position in RF1. Another similar pair is Leu-126 in RF1 and Asp-143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of Arg-213 and Asp-143 with the corresponding RF1 residues will reduce G3 discrimination by RF2, we swapped these residues between RF1 and RF2 by site-directed mutagenesis and characterized their preference for different codons using a competitive peptide release assay. Among these, the R213I mutant of RF2 showed 5-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared with the wild type (WT). In-depth fast kinetic studies revealed that the gain in UAG reading by RF2 R213I is associated with a reduced efficiency of termination on the cognate UAA codon. Our work highlights the notion that stop codon recognition involves complex interactions with multiple residues beyond the PT/SPF motifs. We propose that the R213I mutation in RF2 brings us one step forward toward engineering an omnipotent RF in bacteria, capable of reading all three stop codons.
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http://dx.doi.org/10.1074/jbc.M117.785238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592688PMC
September 2017

Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live .

mBio 2017 06 6;8(3). Epub 2017 Jun 6.

Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA

assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P-mEos2 in live provides the first information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-P exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τ ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τ ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent experiments indicating dissociation of the deacylated tRNA upon translocation. Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.
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http://dx.doi.org/10.1128/mBio.00300-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461404PMC
June 2017

Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.

Nat Microbiol 2017 May 8;2:17068. Epub 2017 May 8.

Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Box 280, Nobels väg 16, 171 77 Stockholm, Sweden.

Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.
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http://dx.doi.org/10.1038/nmicrobiol.2017.68DOI Listing
May 2017

Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.

J Biol Chem 2017 06 18;292(22):9345-9357. Epub 2017 Apr 18.

From the Department of Cell and Molecular Biology, Uppsala University, Uppsala, Box-596, 75124, Sweden,

Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.
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http://dx.doi.org/10.1074/jbc.M116.762096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454114PMC
June 2017

Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein.

J Mol Evol 2017 03 23;84(2-3):69-84. Epub 2017 Feb 23.

School of Biology, Georgia Institute of Technology, 950 Atlantic Drive, Atlanta, GA, 30332, USA.

The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year-old inferred ancestral variant of tufB, an essential gene encoding elongation factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a twofold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2000 generations in the laboratory and examined the adaptive response via fitness assays, whole genome sequencing, proteomics, and biochemical assays. Hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by upregulation of protein production. Our results suggest that an ancient-modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.
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http://dx.doi.org/10.1007/s00239-017-9781-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371648PMC
March 2017

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

Micrococcin P1 - A bactericidal thiopeptide active against Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2016 09 27;100:95-101. Epub 2016 Jul 27.

Department of Molecular Medicine, University of Padova, Padova, Italy. Electronic address:

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.
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http://dx.doi.org/10.1016/j.tube.2016.07.011DOI Listing
September 2016

Molecular mechanism of viomycin inhibition of peptide elongation in bacteria.

Proc Natl Acad Sci U S A 2016 Jan 11;113(4):978-83. Epub 2016 Jan 11.

Department of Cell and Molecular biology, Uppsala University, 75124 Uppsala, Sweden

Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼ 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.
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http://dx.doi.org/10.1073/pnas.1517541113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743798PMC
January 2016

HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

Nat Struct Mol Biol 2015 Nov 12;22(11):906-13. Epub 2015 Oct 12.

Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.

Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.
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http://dx.doi.org/10.1038/nsmb.3103DOI Listing
November 2015