Study of the role of Mg2+ in dsRNA processing mechanism by bacterial RNase III through QM/MM simulations
- 50 Downloads
The ribonuclease III (RNase III) cleaves dsRNA in specific positions generating mature RNAs. RNase III enzymes play important roles in RNA processing, post-transcriptional gene expression, and defense against viral infection. The enzyme’s active site contains Mg2+ ions bound by a network of acidic residues and water molecules, but there is a lack of information about their specific roles. In this work, multiple steered molecular dynamics simulations at QM/MM level were performed to explore the hydrolysis reaction carried out by the enzyme. Free energy profiles modifying the features of the active site are obtained and the role of Mg2+ ions, the solvent molecules and the residues of the active site are discussed in detail. Our results show that Mg2+ ions carry out different roles in the hydrolysis process positioning the substrate for the attack from a coordinated nucleophile and activating it to perform hydrolysis reaction, cleaving the dsRNA backbone in a SN2 substitution. In addition, water molecules present in the active site lower the energy barrier of the process.
KeywordsRNase III QM/MM DFTB Reaction mechanism dsRNA
This research was supported by grants from CONICET and Universidad Nacional de Rosario to D.M.M., S.I.D. has received a fellowship from CONICET. R.R. and D.M.M are members of CONICET. The result presented in this work were obtained using the resources of Centro de Cómputos de Alto Rendimiento (CeCAR), FCEN-UBA.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.Robertson HD, Webster RE, Zinder ND (1968) Purification and properties of ribonuclease III from Escherichia coli. J Biol Chem 213:82–91Google Scholar
- 22.Dasgupta S, Fernandez L, Kameyama L et al (1998) Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coil endoribonuclease RNAse III—the effect of dsRNA binding on gene expression. Mol Microbiol 28:629–640. https://doi.org/10.1046/j.1365-2958.1998.00828.x CrossRefPubMedGoogle Scholar
- 27.Court DL, Gan J, Liang Y-H et al (2013) RNase III: genetics and function; structure and mechanism*. Annu Rev Genet 47:405–431. https://doi.org/10.1146/annurev-genet-110711-155618 CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Case DA, Babin V, Berryman JT et al (2014) AMBER 14. University of California, San FranciscoGoogle Scholar
- 29.Pearlman DA, Case DA, Caldwell JW et al (1995) AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Comp Phys Commun 91:1–41CrossRefGoogle Scholar
- 41.Seabra GDM, Walker RC, Elstner M, et al (2007) Implementation of the SCC-DFTB method for hybrid QM/MM simulations within the Amber molecular dynamics package. J Phys Chem A:5655–5664Google Scholar
- 42.Arrar FM, Boubeta M et al (2018) On the accurate estimation of free energies using the Jarzynski Equality. https://doi.org/10.1002/jcc.25754
- 47.Ramírez CL, Martí MA, Roitberg AE (2016) Steered molecular dynamics methods applied to enzyme mechanism and energetics. Methods in enzymology. Academic Press Inc, London, pp 123–143Google Scholar
- 50.Stevens DR, Hammes-Schiffer S (2018) Exploring the role of the third active site metal ion in DNA polymerase # with QM/MM free energy simulations. https://doi.org/10.1021/jacs.8b05177