Visualizing group II intron dynamics between the first and second steps of splicing

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5’-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy

How effectively bonding evolution theory retrieves and visualizes curly arrows: The cycloaddition reaction of cyclic nitrones

In the present work, the electron density flows involved throughout the progress of the four reaction pathways associated with the intramolecular [3 + 2] cycloaddition of cyclic nitrones Z-1 and E-1 are analyzed using the bonding evolution theory. The present study highlights the nonconcerted nature of the processes, which can be described as taking place in several stages. The first stage consists in the depopulation of the initial C N and C=C double bonds to render the N lone pair and the corresponding C-N and C-C single bonds, and these electronic flows initiate the reactions. The C-C and C-O sigma bond formations take place later on, once the transition states have been overcome. Along the bridged pathways, the C-C bond formation process precedes the O-C bond formation event, although, along the fused paths, the O-C bond formation process occurs first and the formation of the C-C bond is the last electronic flow to take place. Finally, curly arrow representations accounting for the timing of the electron flows are obtained from the bonding evolution theory results

Deuterium Isotope Effects in A:T and A:U Base Pairs: A Computational NMR Study

Abstract:Recent measurements of trans-hydrogen bond deuterium isotope effects (DIEs) on 13C chemical shifts in nucleic acids (Vakonakis, I.; LiWang, A. C. J. Biomol. NMR2004, 29, 65; J. Am. Chem. Soc. 2004, 126, 5688) have led to intriguing results: (i) the DIEs of A:T pairs in DNA are about 5 ppb smaller than those of A:U in RNA and (ii) A:T DIEs vary by as much as 13 ppb among the oligonucleotides. The first observation suggests that inter-base H-bonds in RNA may be stronger than those in DNA, while the second indicates that the conformation of the base pair modulates the transmission of the isotope effect across the hydrogen bond. In an effort at providing a rationale s so far unknown s for the observed DIEs in nucleic acids, density functional theory and hybrid Car Parrinello/molecular mechanical calculations DIEs on nucleosides and nucleotides in the gas phase and in aqueous solution have been performed. The calculations suggest that (i) the DIE in an isolated A:T base pair differs from that in an A:U base pair because of the changes in the magnetic properties caused by the replacement of a methyl group on passing from U to T, (ii) the DIEs depend crucially on the conformation of the base pairs, and (iii) the DIEs are strongly affected by magnetic and electrostatic interactions with the surrounding environment

Molecular Mechanism of Phosphate Steering for DNA Binding, Cleavage Localization, and Substrate Release in Nucleases

Structure-specific endonucleases (SSEs) cleave the DNA substrate in a precise position based on the specific DNA 3D structure. Human flap endonuclease 1 (hFEN1) is a 5′ SSE that prevents DNA instability by processing Okazaki fragment 5′-flaps with remarkable efficiency and selectivity using two-metal-ion catalysis. Recent structural and mutagenesis data of hFEN1 suggest that phosphate steering favors specificity and catalysis. Here, we investigate the phosphate steering mechanism at the atomistic level using microsecond-long molecular dynamics and well-tempered metadynamics simulations of wild-type and mutant systems of hFEN1. We show how positively charged second and third-shell residues operate the phosphate steering mechanism to promote catalysis through (i) substrate recruitment; (ii) precise cleavage localization; and (iii) substrate release, thus actively preventing the off-target incision of the substrate. Importantly, structural comparisons of hFEN1 and other nuclease enzymes suggest that phosphate steering may also serve the structure-based selection of the specific DNA substrate by other 5′ structure-specific nucleases