R Codes to Read a Dna Sequence
Sequence specificity in DNA binding is mainly governed by association
Dominant association
Intuitively, potent binding betwixt two biological macromolecules means that, at whatsoever given time, it is unlikely that they detach. Yet, Marklund et al. evidence that when the lac repressor interacts with Deoxyribonucleic acid, it is primarily the probability of the repressor recognizing its target sequence that dictates the binding force; the time spent in the bound conformation is subordinate. Dissociation will announced slow because the molecules tin undergo many rebinding events after leaving the jump conformation but before separating in space. These results shed new calorie-free on the speed-stability paradox of DNA search kinetics. —DJ
Abstract
Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic clan and dissociation rates of Deoxyribonucleic acid binding proteins. Nosotros tested the model for thousands of different lac operator sequences with a poly peptide bounden microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. Nosotros institute that sequence specificity is mainly governed past the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least i.7 times equally large as the variation in microscopic dissociation rates. Modulating the rate of bounden instead of the rate of dissociation finer reduces the risk of the protein existence retained on nontarget sequences while searching.
Get full access to this article
View all available purchase options and go full admission to this article.
Already a Subscriber?Sign In
Supplementary Materials
This PDF file includes:
Materials and Methods
Supplementary Text
Figs. S1 to S5
Table S1
References (24–38)
Other Supplementary Textile for this manuscript includes the following:
MDAR Reproducibility Checklist
References and Notes
1
Due west. Gilbert, B. Müller-Hill, The lac operator is Deoxyribonucleic acid. Proc. Natl. Acad. Sci. United states of americaA. 58, 2415–2421 (1967).
2
R. Milo, R. Phillips, Cell Biology by the Numbers (Garland Scientific discipline, 2015).
3
A. Grönlund, P. Lötstedt, J. Elf, Transcription factor binding kinetics constrain dissonance suppression via negative feedback. Nat. Commun. 4, 1864 (2013).
4
D. 50. Jones, R. C. Brewster, R. Phillips, Promoter architecture dictates prison cell-to-prison cell variability in cistron expression. Science 346, 1533–1536 (2014).
5
Thousand. Z. Ali, V. Parisutham, S. Choubey, R. C. Brewster, Inherent regulatory asymmetry emanating from network architecture in a prevalent autoregulatory motif. eLife 9, e56517 (2020).
6
Thou. Morrison, G. Razo-Mejia, R. Phillips, Reconciling kinetic and thermodynamic models of bacterial transcription. PLOS Comput. Biol. 17, e1008572 (2021).
vii
P. Hammar, P. Leroy, A. Mahmutovic, Eastward. G. Marklund, O. G. Berg, J. Elf, The lac repressor displays facilitated improvidence in living cells. Science 336, 1595–1598 (2012).
viii
East. Marklund, B. van Oosten, G. Mao, East. Amselem, M. Kipper, A. Sabantsev, A. Emmerich, D. Globisch, X. Zheng, L. C. Lehmann, O. G. Berg, M. Johansson, J. Elf, S. Deindl, Dna surface exploration and operator bypassing during target search. Nature 583, 858–861 (2020).
9
O. Yard. Berg, R. B. Winter, P. H. von Hippel, Diffusion-driven mechanisms of protein translocation on nucleic acids. i. Models and theory. Biochemistry 20, 6929–6948 (1981).
10
M. F. Berger, Thousand. L. Bulyk, Universal protein-bounden microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nat. Protoc. 4, 393–411 (2009).
eleven
T. Siggers, K. H. Duyzend, J. Reddy, S. Khan, M. 50. Bulyk, Non-DNA-binding cofactors raise DNA-binding specificity of a transcriptional regulatory complex. Mol. Syst. Biol. 7, 555 (2011).
12
H. M. Garcia, R. Phillips, Quantitative dissection of the unproblematic repression input–output function. Proc. Natl. Acad. Sci. U.S.A. 108, 12173–12178 (2011).
13
R. C. Brewster, F. M. Weinert, H. G. Garcia, D. Song, M. Rydenfelt, R. Phillips, The transcription factor titration effect dictates level of factor expression. Cell 156, 1312–1323 (2014).
14
P. Hammar, Chiliad. Walldén, D. Fange, F. Persson, O. Baltekin, G. Ullman, P. Leroy, J. Elf, Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation. Nat. Genet. 46, 405–408 (2014).
15
P. C. Blainey, A. M. van Oijen, A. Banerjee, M. L. Verdine, 10. S. Xie, A base of operations-excision Deoxyribonucleic acid-repair protein finds intrahelical lesion bases by fast sliding in contact with Deoxyribonucleic acid. Proc. Natl. Acad. Sci. U.S.A. 103, 5752–5757 (2006).
16
E. A. Boyle, J. O. 50. Andreasson, L. 1000. Chircus, Due south. H. Sternberg, One thousand. J. Wu, C. One thousand. Guegler, J. A. Doudna, W. J. Greenleaf, High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding. Proc. Natl. Acad. Sci. U.s.a.A. 114, 5461–5466 (2017).
17
Thousand. Lewis, Yard. Chang, Due north. C. Horton, M. A. Kercher, H. C. Pace, M. A. Schumacher, R. Chiliad. Brennan, P. Lu, Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247–1254 (1996).
18
J. Chen, S. Alberti, M. Due south. Matthews, Wild-type operator binding and contradistinct cooperativity for inducer bounden of lac repressor dimer mutant R3. J. Biol. Chem. 269, 12482–12487 (1994).
19
S. L. Laiken, C. A. Gross, P. H. Von Hippel, Equilibrium and kinetic studies of Escherichia coli lac repressor-inducer interactions. J. Mol. Biol. 66, 143–155 (1972).
20
A. Poddar, One thousand. S. Azam, T. Kayikcioglu, Chiliad. Bobrovskyy, J. Zhang, X. Ma, P. Labhsetwar, J. Fei, D. Singh, Z. Luthey-Schulten, C. K. Vanderpool, T. Ha, Effects of private base-pairs on in vivo target search and devastation kinetics of bacterial small RNA. Nat. Commun. 12, 874 (2021).
21
N. F. Dupuis, E. D. Holmstrom, D. J. Nesbitt, Single-molecule kinetics reveal cation-promoted DNA duplex germination through ordering of single-stranded helices. Biophys. J. 105, 756–766 (2013).
22
S. Bonilla, C. Limouse, Due north. Bisaria, Chiliad. Gebala, H. Mabuchi, D. Herschlag, Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway. J. Am. Chem. Soc. 139, 18576–18589 (2017).
23
E. Marklund, G. Mao, J. Yuan, S. Zikrin, Eastward. Abdurakhmanov, S. Deindl, J. Elf, Data and code for: Sequence specificity in DNA bounden is mainly governed past association (Version ane.0). SciLifeLab (2021); https://doi.org/10.17044/scilifelab.17099687.
24
K. Kipper, N. Eremina, E. Marklund, S. Tubasum, Chiliad. Mao, 50. C. Lehmann, J. Elf, Due south. Deindl, Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI. PLOS ONE thirteen, e0198416 (2018).
25
A. D. Edelstein, Grand. A. Tsuchida, Northward. Amodaj, H. Pinkard, R. D. Vale, Northward. Stuurman, Avant-garde methods of microscope command using μManager software. J. Biol. Methods one, e10 (2014).
26
S. Deindl, X. Zhuang, Monitoring conformational dynamics with single-molecule fluorescence energy transfer: Applications in nucleosome remodeling. Methods Enzymol. 513, 59–86 (2012).
27
A. Sabantsev, R. F. Levendosky, X. Zhuang, G. D. Bowman, S. Deindl, Direct observation of coordinated Dna movements on the nucleosome during chromatin remodelling. Nat. Commun. x, 1720 (2019).
28
J.-C. Olivo-Marin, Extraction of spots in biological images using multiscale products. Pattern Recognit. 35, 1989–1996 (2002).
29
B. G. Sadler, A. Swami, Assay of multiscale products for step detection and estimation. IEEE Trans. Inf. Theory 45, 1043–1051 (1999).
xxx
D. Garcia, Robust smoothing of gridded data in i and college dimensions with missing values. Comput. Stat. Data Anal. 54, 1167–1178 (2010).
31
M. Lindén, V. Ćurić, A. Boucharin, D. Fange, J. Elf, Simulated single molecule microscopy with SMeagol. Bioinformatics 32, 2394–2395 (2016).
32
S. H. Sternberg, S. Redding, M. Jinek, East. C. Greene, J. A. Doudna, DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).
33
X. Wu, D. A. Scott, A. J. Kriz, A. C. Chiu, P. D. Hsu, D. B. Dadon, A. Due west. Cheng, A. E. Trevino, S. Konermann, S. Chen, R. Jaenisch, F. Zhang, P. A. Precipitous, Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).
34
Thousand. D. Szczelkun, Yard. S. Tikhomirova, T. Sinkunas, G. Gasiunas, T. Karvelis, P. Pschera, V. Siksnys, R. Seidel, Direct ascertainment of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl. Acad. Sci. United statesA. 111, 9798–9803 (2014).
35
Thou. Klein, B. Eslami-Mossallam, D. G. Arroyo, Grand. Depken, Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules. Cell Rep. 22, 1413–1423 (2018).
36
R. B. Winter, P. H. von Hippel, Improvidence-driven mechanisms of poly peptide translocation on nucleic acids. 2. The Escherichia coli repressor—operator interaction: Equilibrium measurements. Biochemistry twenty, 6948–6960 (1981).
37
D. Yang, A. Singh, H. Wu, R. Kroe-Barrett, Comparison of biosensor platforms in the evaluation of high affinity antibiotic-antigen binding kinetics. Anal. Biochem. 508, 78–96 (2016).
38
J. Elf, K.-W. Li, X. S. Xie, Probing transcription gene dynamics at the unmarried-molecule level in a living cell. Science 316, 1191–1194 (2007).
Data & Authors
Information
Published In
Science
Volume 375 | Upshot 6579
28 January 2022
Copyright
Copyright © 2022 The Authors, some rights reserved; sectional licensee American Association for the Advocacy of Science. No claim to original U.South. Regime Works.
Submission history
Received: 25 January 2021
Accustomed: 21 Dec 2021
Published in print: 28 January 2022
Acknowledgments
We give thanks O. Berg, 1000. Ehrenberg, H. Danielson, J. Wiktor, K. Lüking, D. Fange, I. Barkefors, and D. Jones for discussions.
Funding: This inquiry was supported by the Knut and Alice Wallenberg Foundation (2016.0077 and 2019.0439 to J.Due east.; 2019.0306 to Southward.D.), the Swedish Research Council (2016-06213 to J.E.; 2020-06459 to E.M.), the European Research Council (Starting Grant, 714068 to S.D.; Advanced Grant, 885360 to J.E.), the eSSENCE e-scientific discipline initiative and the Swedish National Infrastructure for Computing (SNIC) at UPPMAX.
Author contributions: J.E. and E.G. conceived the study; E.M. derived models and equations; S.D., E.Grand., and M.G. designed the single-molecule experiments; M.Grand. performed the single-molecule experiments; Eastward.1000. analyzed the unmarried-molecule data; J.Y., E.1000., and J.E. designed the PBM experiments; J.Y. performed the PBM experiments; J.Y. and Due south.Z. analyzed the PBM experiments; E.M. and EA designed, EA performed, and E.Chiliad. analyzed the SPR experiments; E.1000., J.E., and S.D. interpreted the results and wrote the paper, with input from all authors.
Competing interests: The authors declare no competing interests.
Information and materials availability: All raw data and analysis codes are available at the SciLifeLab Repository (23).
Authors
Funding Information
KAW: 2016.0077
KAW: 2019.0439
KAW: 2019.0306
Metrics & Citations
Metrics
Article Usage
Altmetrics
Citations
Consign commendation
Select the format you desire to consign the citation of this publication.
View Options
Get Access
View options
PDF format
Download this article every bit a PDF file
Download PDF
Media
Figures
Multimedia
Tables
Source: https://www.science.org/doi/10.1126/science.abg7427
0 Response to "R Codes to Read a Dna Sequence"
Post a Comment