Efficient fidelity control by stepwise nucleotide selection in polymerase elongation
Polymerases select nucleotides according to a template before incorporating them for chemical synthesis during gene replication or transcription. Efficient selection to achieve sufficiently high fidelity and speed is essential for polymerase function. Due to multiple kinetic steps detected in a polymerase elongation cycle, there exist multiple selection checkpoints to allow different strategies of fidelity control. In our current work, we examined step-by-step selections in an elongation cycle that have conformational transition rates tuned one at a time, with a controlled differentiation free energy between the right and wrong nucleotides at each checkpoint. The elongation is sustained at non-equilibrium steady state with constant free energy input and heat dissipation. It is found that a selection checkpoint in the later stage of a reaction path has less capability for error reduction. Hence, early selection is essential to achieve an efficient fidelity control. In particular, for an intermediate state, the selection through the forward transition inhibition has the same capacity for error reduction as the selection through the backward rejection. As with respect to the elongation speed, an initial screening is indispensible for maintaining high speed, as the wrong nucleotides can be removed quickly and replaced by the right ones at the entry. Overall, the elongation error rate can be repeatedly reduced through multiple selection checkpoints. This study provides a theoretical framework to guide more detailed structural dynamics studies, and to support rational redesign of related enzymes and devices.
-  Buc, H., and T. Strick, editors. 2009. RNA polymerase as molecular motors. The Royal Society of Chemistry, Cambridge, UK.
-  Bar-Nahum, G., V. Epshtein, A. E. Ruckenstein, R. Rafikov, A. Mustaev, and E. Nudler. 2005. A Ratchet Mechanism of TranscriptionElongation and Its Control. Cell 120:183-193.
-  Abbondanzieri, E. A., W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block. 2005. Direct observation of base-pairstepping by RNA polymerase. Nature 438:460-465.
-  Thomen, P., P. J. Lopez, and F. Heslot. 2005. Unravelling the Mechanism of RNA-Polymerase Forward Motion by Using MechanicalForce. Physical Review Letters 94:128102.[PubMed][Crossref]
-  Guo, Q., and R. Sousa. 2006. Translocation by T7 RNA Polymerase: A Sensitively Poised Brownian Ratchet. Journal ofMolecularBiology 358:241-254.
-  Wang, H., and G. Oster. 2002. Ratchets, power strokes, and molecular motors. Applied Physics A 75:315-323.
-  Yu, J., and G. Oster. 2012. A Small Post-Translocation Energy Bias Aids Nucleotide Selection in T7 RNA Polymerase Transcription.Biophysical Journal 102:532-541.[WoS][Crossref]
-  Johnson, K. A. 1993. Conformational coupling in DNA polymerase fidelity. Annual Review of Biochemistry 62:685-713.[Crossref]
-  Schlick, T., K. Arora, W. A. Beard, and S. H. Wilson. 2012. Perspective: pre-chemistry conformational changes in DNA polymerasemechanisms. Theoretical Chemistry Accounts 131:1287.[WoS]
-  McCulloch, S. D., and T. A. Kunkel. 2008. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesispolymerases. Cell Research 18:148-161.[WoS][Crossref][PubMed]
-  Joyce, C. M. 1997. Choosing the right sugar: How polymerase select a nucleotide substrate. Proceedings of the NationalAcademy of Sciences of the United States of America 94:1619-1622.
-  Joyce, C. M., and S. J. Benkovic. 2004. DNA polymerase fidelity: kinetics, structure, and checkpoints. Biochemistry 43:14317-14324.[WoS]
-  Sydow, J. F., and P. Cramer. 2009. RNA polymerase fidelity and transcriptional proofreading. Current Opinion in StructuralBiology 19:732-739.[Crossref][WoS]
-  Johnson, K. A. 2010. The kinetic and chemical mechanism of high-fidelity DNA polymerases. Biochimica et Biophysica Acta1804:1041-1048.[WoS]
-  Hopfield, J. J. 1974. Kinetic Proofreading: A New Mechanism for Reducing Errors in Biosynthetic Processes Requiring HighSpecificity. Proceedings of the National Academy of Sciences of the United States of America 71:4135-4139.
-  Ninio, J. 1975. Kinetic amplification of enzyme discrimination. Biochimie 57:587–595.
-  Ehrenberg, M., and C. Blomberg. 1980. Thermodynamic constraints on kinetic proofreading in biosynthetic pathways. BiophysicalJournal 31:333-358.[PubMed][Crossref]
-  Blanchard, S., R. Gonzalez, H. Kim, S. Chu, and J. Puglisi. 2004. tRNA selection and kinetic proofreading in translation.Nature structural & molecular biology 11:1008-1014.
-  Johansson, M., J. Zhang, and M. Ehrenberg. 2012. Genetic code translation displays a linear trade-off between eflciency andaccuracy of tRNA Selection. Proceedings of the National Academy of Sciences of the United States of America 109:131-136.[WoS]
-  Galburt, E. A., S.W. Grill, A.Wiedmann, L. Lubkowska, J. Choy, E. Nogales, M. Kashlev, and C. Bustamante. 2007. Backtrackingdetermines the force sensitivity of RNAP II in a factor-dependent manner. Nature 446:820-823.[WoS]
-  Herbert, K. M., W. J. Greenleaf, and S. M. Block. 2008. Single-Molecule Studies of RNA Polymerase: Motoring Along. AnnualReview of Biochemistry 77.[WoS]
-  Tadigotla, V. R., D. O. Maoileidigh, A. M. Sengupta, V. Espshtein, R. H. Ebright, E. Nudler, and A. E. Ruckenstein. 2006.Thermodynamic and kinetic modeling of transcriptional pausing. Proceedings of the National Academy of Sciences of theUnited States of America 103:4439-4444.
-  Voliotis, M., N. Cohen, C. Molina-Paris, and T. B. Liverpool. 2009. Backtracking and Proofreading in DNATranscription.Physicsal Reveiw Letters 102:258101.[Crossref]
-  Depken, M. D., E. A. Galburt, and S. W. Grill. 2009. The Origin of Short Transcriptional Pauses. Biophysical Journal 96:2189-2193.[WoS][Crossref][PubMed]
-  Violiotis, M., N. Cohen, C. Molina-Paris, and L. T.B. 2012. Proofreading of misincorporated nucleotides in DNA transcription.Physical Biology 93:036007.[Crossref]
-  Yuzenkova, Y., A. Bochkareva, V. R. Tadigotla, M. Roghanian, S. Zorov, K. Severinov, and N. Zenkin. 2010. Stepwise mechanismfor transcription fidelity. BMC Biology 8:54.[Crossref][PubMed][WoS]
-  Cady, F., and H. Qian. 2009. Open-system thermodynamic analysis of DNA polymerase fidelity. Physical Biology 6:036011.[PubMed][WoS][Crossref]
-  Andrieux, D., and P. Gaspard. 2008. Nonequilibrium generation of information in copolymerization processes. Proceedingsof the National Academy of Sciences of the United States of America 105:9516-9521.[WoS]
-  Ehrenberg, M., and C. G. Kurland. 1984. Costs of accuracy determined by a maximal growth rate constraint. Quarterly Reviewsof Biophysics 17:45-82.[Crossref]
-  Kurland, C. G., and M. Ehrenberg. 1984. Optimization of translation accuracy. Progress in Nucleic Acid Research and MolecularBiology 31:191-219.
-  Anand, V. S., and S. S. Patel. 2006. Transient State Kinetics of Transcription Elongation by T7 RNA Polymerase. The Journalof Biological Chemistry 281:35677-35685.
-  Huang, J., L. G. Brieba, and R. Sousa. 2000. Misincorporation by Wild-Type and Mutant T7 RNA Polymerases: Identificationof Interactions That Reduce Misincorporation Rates by Stabilizing the Catalytically Incompetent Open Conformation.Biochemistry 39:11571-11580.[Crossref]
-  Yin, Y.W., and T. A. Steitz. 2004. The Structural Mechanism of Translocation and Helicase Activity in T7 RNA Polymerase. Cell116:393-404.
-  . Temiakov, D., V. Patlan, M. Anikin, W. T. McAllister, S. Yokoyama, and D. G. Vassylyev. 2004. Structural Basis for SubstrateSelection by T7 RNA Polymerase. Cell 116:381-391.
-  Sousa, R., and R. Padilla. 1995. A mutant T7 RNA polymerase as a DNA polymerase. The EMBO Journal 14:4609-4621.
-  Petruska, J., L. C. Sowers, and M. F. Goodman. 1986. Comparison of nucleotide interactions in water, proteins, and vacuum:Model for DNA polymerase fidelity. Proceedings of the National Academy of Sciences of the United States of America 83:1559-1562.
-  Feig, M., and Z. F. Burton. 2010. RNA Polymerase II with Open and Closed Trigger Loops: Active Site Dynamics and NucleicAcid Translocation. Biophysical Journal 99:2577-2586.[WoS]
-  Dangkulwanich, M., T. Ishibashi, S. Liu, M. L. Kireeva, L. Lubkowska, M. Kashlev, and C. J. Bustamante. 2013. Completedissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. eLife2:e00971.[WoS]
-  Thomen, P., P. J. Lopez, U. Bockelmann, J. Guillerez, M. Dreyfus, and F. Heslot. 2008. T7 RNA Polymerase Studied by ForceMeasurements Varying Cofactor Concentration. Biophysical Journal 95:2423-2433.[WoS][Crossref]
-  Bai, L., T. J. Santangelo, and M. D. Wang. 2006. Single-Molecule Analysis of RNA Polymerase Transcription. Annual Reviewof Biophysics and Biomolecular Structure 35:343-360. [Crossref]