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Liao, Q., Lüking, M., Krueger, D. M., Deindl, S., Elf, J., Kasson, P. M. & Kamerlin, S. C. (2019). Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition. Journal of Physical Chemistry B, 123(17), 3576-3590
Open this publication in new window or tab >>Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition
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2019 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 123, no 17, p. 3576-3590Article in journal (Refereed) Published
Abstract [en]

Recent years have witnessed an explosion of interest in computational studies of DNA binding proteins, including both coarse grained and atomistic simulations of transcription factor-DNA recognition, to understand how these transcription factors recognize their binding sites on the DNA with such exquisite specificity. The present study performs microsecond time scale all-atom simulations of the dimeric form of the lactose repressor (Lad), both in the absence of any DNA and in the presence of both specific and nonspecific complexes, considering three different DNA sequences. We examine, specifically, the conformational differences between specific and nonspecific protein DNA interactions, as well as the behavior of the helix-turn-helix motif of Lad when interacting with the DNA. Our simulations suggest that stable Lad binding occurs primarily to bent A-form DNA, with a loss of Lad conformational entropy and optimization of correlated conformational equilibria across the protein. In addition, binding to the specific operator sequence involves a slightly larger number of stabilizing DNA protein hydrogen bonds (in comparison to nonspecific complexes), which may account for the experimentally observed specificity for this operator. In doing so, our simulations provide a detailed atomistic description of potential structural drivers for LacI selectivity.

National Category
Physical Chemistry Biophysics
Identifiers
urn:nbn:se:uu:diva-384077 (URN)10.1021/acs.jpcb.8b12363 (DOI)000466989000003 ()30952192 (PubMedID)
Funder
Swedish Research Council, 2016-06213Knut and Alice Wallenberg Foundation, KAW 2016.0077
Available from: 2019-05-28 Created: 2019-05-28 Last updated: 2019-05-28Bibliographically approved
Elf, J. & Barkefors, I. (2019). Single-Molecule Kinetics in Living Cells. Annual Review of Biochemistry, 88, 635-659
Open this publication in new window or tab >>Single-Molecule Kinetics in Living Cells
2019 (English)In: Annual Review of Biochemistry, ISSN 0066-4154, E-ISSN 1545-4509, Vol. 88, p. 635-659Article, review/survey (Refereed) Published
Abstract [en]

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

Place, publisher, year, edition, pages
ANNUAL REVIEWS, 2019
Keywords
single-molecule microscopy, single-molecule kinetics, fluorescence labeling, single-molecule tracking, spot localization, simulated microscopy
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390235 (URN)10.1146/annurev-biochem-013118-110801 (DOI)000472818600025 ()30359080 (PubMedID)978-0-8243-0888-9 (ISBN)
Available from: 2019-08-08 Created: 2019-08-08 Last updated: 2019-08-08Bibliographically approved
Jones, D. & Elf, J. (2018). Bursting onto the scene?: Exploring stochastic mRNA production in bacteria. Current Opinion in Microbiology, 45, 124-130
Open this publication in new window or tab >>Bursting onto the scene?: Exploring stochastic mRNA production in bacteria
2018 (English)In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 45, p. 124-130Article, review/survey (Refereed) Published
Abstract [en]

Recent large-scale measurements of gene expression variability (or noise) in E. coli have led to the unexpected conclusion that the variability is in large part dictated by and increasing with the mean level of expression. Here we review the evidence for this apparent universal trend in variability, as well as for the related idea that transcription is fundamentally bursty. We examine recently proposed mechanisms for burstiness and universality and argue that they do not explain important features of observed data. Finally, we discuss potential limitations and pitfalls in the interpretation of experimental measurements of cell-to-cell variability.

Place, publisher, year, edition, pages
CURRENT BIOLOGY LTD, 2018
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-375611 (URN)10.1016/j.mib.2018.04.001 (DOI)000454972700019 ()29705632 (PubMedID)
Funder
Swedish Research Council, VR 642-2013-7841Swedish Research Council, VR 621-2012-4027EU, European Research Council, ERC-2013-CoG/616047EU, Horizon 2020, MSCA-IF-2015-704206
Available from: 2019-01-31 Created: 2019-01-31 Last updated: 2019-01-31Bibliographically approved
Ghosh, A., Baltekin, Ö., Wäneskog, M., Elkhalifa, D., Larsson, D., Elf, J. & Koskiniemi, S. (2018). Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations. EMBO Journal, 37(9), Article ID UNSP e98026.
Open this publication in new window or tab >>Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations
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2018 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed) Published
Abstract [en]

Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
bet-hedging, contact-dependent growth inhibition, persisters, toxin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356400 (URN)10.15252/embj.201798026 (DOI)000431279400003 ()29572241 (PubMedID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilEU, European Research Council
Note

Anirban Ghosh and Özden Baltekin contributed equally to this work.

Available from: 2018-07-25 Created: 2018-07-25 Last updated: 2019-05-13Bibliographically approved
Kipper, K., Eremina, N., Marklund, E., Tubasum, S., Mao, G., Lehmann, L. C., . . . Deindl, S. (2018). Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI. PLoS ONE, 13(6), Article ID e0198416.
Open this publication in new window or tab >>Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI
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2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed) Published
Abstract [en]

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-358704 (URN)10.1371/journal.pone.0198416 (DOI)000433900800119 ()29856839 (PubMedID)
Funder
EU, European Research Council, 714068Swedish Research Council, VR 2015-04568Knut and Alice Wallenberg Foundation, WAF 2014.0183EU, European Research Council
Note

De tre första författarna delar förstaförfattarskapet.

Available from: 2018-08-31 Created: 2018-08-31 Last updated: 2018-08-31Bibliographically approved
Volkov, I., Lindén, M., Aguirre, J., Ieong, K.-W., Metelev, M., Elf, J. & Johansson, M. (2018). tRNA tracking for direct measurements of protein synthesis kinetics in live cells. Nature Chemical Biology, 14(6), 618-626
Open this publication in new window or tab >>tRNA tracking for direct measurements of protein synthesis kinetics in live cells
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2018 (English)In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed) Published
Abstract [en]

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-359663 (URN)10.1038/s41589-018-0063-y (DOI)000435445100019 ()29769736 (PubMedID)
Funder
Swedish Research Council, 2015-04111EU, European Research Council, ERC-2013-CoG 616047 SMILEKnut and Alice Wallenberg FoundationWenner-Gren FoundationsCarl Tryggers foundation , CTS 15:243
Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2018-09-05Bibliographically approved
Lindén, M. & Elf, J. (2018). Variational Algorithms for Analyzing Noisy Multistate Diffusion Trajectories. Paper presented at Biophysical-Society Thematic Meeting on Single-Cell Biophysics - Mearurement, Modulation, and Modeling, JUN, 2017, Natl Taiwan Univ, Acad Sinica, Inst Atom & Mol Sci, Taipei, TAIWAN. Biophysical Journal, 115(2), 276-282
Open this publication in new window or tab >>Variational Algorithms for Analyzing Noisy Multistate Diffusion Trajectories
2018 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 115, no 2, p. 276-282Article in journal (Refereed) Published
Abstract [en]

Single-particle tracking offers a noninvasive high-resolution probe of biomolecular reactions inside living cells. However, efficient data analysis methods that correctly account for various noise sources are needed to realize the full quantitative potential of the method. We report algorithms for hidden Markov-based analysis of single-particle tracking data, which incorporate most sources of experimental noise, including heterogeneous localization errors and missing positions. Compared to previous implementations, the algorithms offer significant speedups, support for a wider range of inference methods, and a simple user interface. This will enable more advanced and exploratory quantitative analysis of single-particle tracking data.

Place, publisher, year, edition, pages
CELL PRESS, 2018
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-361691 (URN)10.1016/j.bpj.2018.05.027 (DOI)000438958800014 ()29937205 (PubMedID)
Conference
Biophysical-Society Thematic Meeting on Single-Cell Biophysics - Mearurement, Modulation, and Modeling, JUN, 2017, Natl Taiwan Univ, Acad Sinica, Inst Atom & Mol Sci, Taipei, TAIWAN
Funder
Knut and Alice Wallenberg FoundationEU, European Research Council, ERC-2013-CoG 616047 SMILE
Available from: 2018-10-03 Created: 2018-10-03 Last updated: 2018-10-03Bibliographically approved
Baltekin, Ö., Boucharin, A., Tano, E., Andersson, D. I. & Elf, J. (2017). Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging. Proceedings of the National Academy of Sciences of the United States of America, 114(34), 9170-9175
Open this publication in new window or tab >>Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 34, p. 9170-9175Article in journal (Refereed) Published
Abstract [en]

The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (10(4) cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.

Keywords
point of care, UTI, AST, antibiotic, resistance, microfluidic
National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-333967 (URN)10.1073/pnas.1708558114 (DOI)000408095300072 ()
Available from: 2017-12-13 Created: 2017-12-13 Last updated: 2018-01-13Bibliographically approved
Kipper, K., Lundius, E. G., Ćurić, V., Nikic, I., Wiessler, M., Lemke, E. A. & Elf, J. (2017). Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli. ACS Photonics, 6(2), 233-255
Open this publication in new window or tab >>Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli
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2017 (English)In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 2, p. 233-255Article in journal (Refereed) Published
Abstract [en]

Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
Keywords
noncanonical amino acid, tetrazine, recoded E. coli, in vivo fluorescence labeling, OmpC, single particle tracking
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-320513 (URN)10.1021/acssynbio.6b00138 (DOI)000394736400009 ()27775882 (PubMedID)
Funder
EU, European Research CouncilSwedish Research CouncilKnut and Alice Wallenberg FoundationGerman Research Foundation (DFG), SPP1623
Available from: 2017-04-20 Created: 2017-04-20 Last updated: 2018-06-14Bibliographically approved
Doudna, J., Bar-Ziv, R., Elf, J., Noireaux, V., Berro, J., Saiz, L., . . . Fordyce, P. (2017). How Will Kinetics and Thermodynamics Inform Our Future Efforts to Understand and Build Biological Systems?. CELL SYSTEMS, 4(2), 144-146
Open this publication in new window or tab >>How Will Kinetics and Thermodynamics Inform Our Future Efforts to Understand and Build Biological Systems?
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2017 (English)In: CELL SYSTEMS, ISSN 2405-4712, Vol. 4, no 2, p. 144-146Article in journal (Refereed) Published
Place, publisher, year, edition, pages
CELL PRESS, 2017
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:uu:diva-321012 (URN)000395786100003 ()
Available from: 2017-04-28 Created: 2017-04-28 Last updated: 2017-04-28Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-5522-1810

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