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Mandava, Chandra SekharORCID iD iconorcid.org/0000-0002-3028-3270
Publications (10 of 10) Show all publications
Holm, M., Mandava, C. S., Ehrenberg, M. & Sanyal, S. (2019). The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation. eLIFE, 8, Article ID e46124.
Open this publication in new window or tab >>The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation
2019 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e46124Article in journal (Refereed) Published
Abstract [en]

Applying pre-steady state kinetics to an Escherichia-coli-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.

Place, publisher, year, edition, pages
ELIFE SCIENCES PUBLICATIONS LTD, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390687 (URN)10.7554/eLife.46124 (DOI)000473013700001 ()31172942 (PubMedID)
Funder
Swedish Research Council, 2018-05498Swedish Research Council, 2016-06264Knut and Alice Wallenberg Foundation, KAW 2011.0081Knut and Alice Wallenberg Foundation, KAW 2017.0055Carl Tryggers foundation , CTS 18: 338Wenner-Gren Foundations, UPD2017-0238
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
Ge, X., Mandava, C. S., Lind, C., Åqvist, J. & Sanyal, S. (2018). Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association. Proceedings of the National Academy of Sciences of the United States of America, 115(18), 4649-4654
Open this publication in new window or tab >>Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed) Published
Abstract [en]

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 Escherichia coli 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.

Keywords
protein synthesis, ribosomal protein L7/12, protein-protein interaction, ribosome, translation initiation
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-350185 (URN)10.1073/pnas.1802001115 (DOI)000431119600050 ()29686090 (PubMedID)
Funder
Swedish Research Council, 2014-4423; 2016-06264Knut and Alice Wallenberg Foundation, 2011.0081VINNOVA, 2013-8778
Available from: 2018-05-07 Created: 2018-05-07 Last updated: 2018-07-13Bibliographically approved
Fislage, M., Zhang, J., Brown, Z. P., Mandava, C. S., Sanyal, S., Ehrenberg, M. & Frank, J. (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-Tu(H84A). Nucleic Acids Research, 46(11), 5861-5874
Open this publication in new window or tab >>Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-Tu(H84A)
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2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, p. 5861-5874Article in journal (Refereed) Published
Abstract [en]

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 themonitoring 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.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-361119 (URN)10.1093/nar/gky346 (DOI)000438362400042 ()29733411 (PubMedID)
Funder
Swedish Research Council, 2013-8778Swedish Research Council, 2014-4423Swedish Research Council, 2016-06264Swedish Research Council, 2017-00230Knut and Alice Wallenberg Foundation, KAW 2011.0081NIH (National Institute of Health), R01 GM29169
Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-09-20Bibliographically approved
Chan, S., Frasch, A., Mandava, C. S., Ch'ng, J.-H., del Pilar Quintana, M., Vesterlund, M., . . . Wahlgren, M. (2017). Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor. Nature Microbiology, 2(7), Article ID 17068.
Open this publication in new window or tab >>Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor
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2017 (English)In: Nature Microbiology, E-ISSN 2058-5276, Vol. 2, no 7, article id 17068Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-333532 (URN)10.1038/nmicrobiol.2017.68 (DOI)000406925300009 ()28481333 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2011.0081Swedish Research Council, VR/2012-2014/521-2011-3377 VR 2013-8778 2014-4423 2016-06264
Available from: 2017-11-14 Created: 2017-11-14 Last updated: 2018-04-18Bibliographically approved
Degiacomi, G., Personne, Y., Mondesert, G., Ge, X., Mandava, C. S., Hartkoorn, R. C., . . . Manganelli, R. (2016). Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis. Tuberculosis, 100, 95-101
Open this publication in new window or tab >>Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis
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2016 (English)In: Tuberculosis, ISSN 1472-9792, E-ISSN 1873-281X, Vol. 100, p. 95-101Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Tuberculosis, Drug development, Translation, Thiopeptides
National Category
Cell and Molecular Biology Respiratory Medicine and Allergy
Identifiers
urn:nbn:se:uu:diva-307870 (URN)10.1016/j.tube.2016.07.011 (DOI)000382201400013 ()27553416 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 260872Swedish Research Council, 2014-4423
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2019-11-08Bibliographically approved
Koripella, R. K., Holm, M., Dourado, D., Mandava, C. S., Flores, S. & Sanyal, S. (2015). A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate. Scientific Reports, 5, Article ID 12970.
Open this publication in new window or tab >>A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12970Article in journal (Refereed) Published
Abstract [en]

Elongation factor G (EF-G), a translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the apex of switch-II, which has been implicated in GTPase activation and GTP hydrolysis. While H91A, H91R and H91E mutants showed different degrees of defect in ribosome associated GTP hydrolysis, H91Q behaved like the WT. However, all these mutants, including H91Q, are much more defective in inorganic phosphate (Pi) release, thereby suggesting that H91 facilitates Pi release. In crystal structures of the ribosome bound EF-G center dot GTP a tight coupling between H91 and the gamma-phosphate of GTP can be seen. Following GTP hydrolysis, H91 flips similar to 140 degrees in the opposite direction, probably with Pi still coupled to it. This, we suggest, promotes Pi to detach from GDP and reach the inter-domain space of EF-G, which constitutes an exit path for the Pi. Molecular dynamics simulations are consistent with this hypothesis and demonstrate a vital role of an Mg2+ ion in the process.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-261233 (URN)10.1038/srep12970 (DOI)000359373400001 ()
Funder
Swedish Research Council, 2011-6088 2014-4423 2008-6593Knut and Alice Wallenberg Foundation, KAW 2011.0081
Available from: 2015-09-07 Created: 2015-08-31 Last updated: 2017-12-04Bibliographically approved
Zhang, Y., Mandava, C. S., Cao, W., Li, X., Zhang, D., Li, N., . . . Gao, N. (2015). HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions. Nature Structural & Molecular Biology, 22(11), 906-913
Open this publication in new window or tab >>HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions
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2015 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 22, no 11, p. 906-913Article in journal (Refereed) Published
Abstract [en]

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 coil 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.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-268420 (URN)10.1038/nsmb.3103 (DOI)000364273800014 ()26458047 (PubMedID)
Funder
Swedish Research Council, 2010-2619Swedish Research Council, 2011-6088Swedish Research Council, 2014-4423Swedish Research Council, 2008-6593Knut and Alice Wallenberg Foundation, KAW 2011.0081
Available from: 2015-12-06 Created: 2015-12-04 Last updated: 2017-12-01
Feng, B., Mandava, C. S., Guo, Q., Wang, J., Cao, W., Li, N., . . . Gao, N. (2014). Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase. PLoS biology, 12(5), e1001866
Open this publication in new window or tab >>Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase
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2014 (English)In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, no 5, p. e1001866-Article in journal (Refereed) Published
Abstract [en]

Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S? ObgE? GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p) ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p) ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-228493 (URN)10.1371/journal.pbio.1001866 (DOI)000336969200016 ()
Available from: 2014-07-15 Created: 2014-07-15 Last updated: 2017-12-05Bibliographically approved
Mandava, C. S., Peisker, K., Ederth, J., Kumar, R., Ge, X., Szaflarski, W. & Sanyal, S. (2012). Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G. Nucleic Acids Research, 40(5), 2054-2064
Open this publication in new window or tab >>Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 5, p. 2054-2064Article in journal (Refereed) Published
Abstract [en]

The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

Keywords
ribosome, translation initiation, L12, IF2, subunit association, protein synthesis
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-157694 (URN)10.1093/nar/gkr1031 (DOI)000302019900022 ()22102582 (PubMedID)
Available from: 2012-03-13 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved
Guo, X., Peisker, K., Bäckbro, K., Chen, Y., Koripella, R. K., Mandava, C. S., . . . Selmer, M. (2012). Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling. Open Biology, 2, 120016
Open this publication in new window or tab >>Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
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2012 (English)In: Open Biology, ISSN 2046-2441, Vol. 2, p. 120016-Article in journal (Refereed) Published
Abstract [en]

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 angstrom crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G-ribosome complex.

Keywords
FusB, elongation factor G, fusidic acid, antibiotic resistance
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-181147 (URN)10.1098/rsob.120016 (DOI)000307111800002 ()
Available from: 2012-09-18 Created: 2012-09-17 Last updated: 2016-01-13Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-3028-3270

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