Pre-mRNA Splicing: An Evolutionary Computational Journey from Ribozymes to Spliceosome

The intron\u2013exon organization of the genes is nowadays taken for granted and constitutes a fully established theory. DNA protein-coding sequences (exons) are not contiguous but rather separated by silent intervening fragments (introns), which must be removed in a process called pre-mRNA splicing. However, this fragmented composition of the eukaryotic genome has ancient origins. It appears that, during the initial stages of eukaryotic evolution, group II introns, i.e. self-splicing catalytic ribozymes, invaded the eukaryotic genome via the endosymbiosis of an alpha-proteobacterium in an archaeal host. At a later time, they split into the inert spliceosomal introns and the catalytically active small nuclear (sn)RNAs, which, together with additional splicing factors, gave rise to the eukaryotic spliceosome. This marked the transition from the autocatalytic splicing, mediated by ribozymes (RNA filaments endowed with an intrinsic catalytic activity) to splicing mediated by a protein-RNA machinery, the spliceosome. In the present thesis, the evolutionary relationship between group II introns and the spliceosome is retraced from a computational perspective by means of classical molecular dynamics simulations (MD), quantum mechanics calculations (QM) and combined quantum-classical simulations (QM/MM). The splicing process of these two different \u2013 but mechanistically related \u2013 large and sophisticated biomolecules is investigated with the aim of deciphering the reactivity and the structural properties from a computational point of view, with a focus on the role played by the Mg2+ ions as splicing cofactors. In Chapter 2, the importance of Mg2+ ions in the RNA biology is introduced. Not only they participate to the catalysis, but also represent essential structural and functional elements for RNA filaments. Moreover, the structural and molecular biology of group II intron ribozymes and the spliceosome machinery are widely discussed with a focus on their evolutionary links. Chapter 3 consists of a brief review of all the computational techniques employed in this thesis, from classical MD to QM and QM/MM simulations and enhanced sampling methods aimed at reconstructing the free energy of a process. Chapter 4 is entirely dedicated to the splicing mechanism promoted by group II intron ribozymes, representing the starting point of the evolutionary journey. In this chapter, a QM/MM study of the molecular mechanism of group II introns first-step hydrolytic splicing is presented, unveiling an RNA-adapted Steitz and Steitz\u2019s two-Mg2+-ion dissociative catalysis which differs from the one observed in protein enzymes. Chapter 5 is focused on Mg2+ ions, which are the natural cofactors of splicing, both in group II introns and in the spliceosome. Mg2+/RNA interplay is here addressed using a group II intron as a prototype of a large RNA molecule binding Mg2+. The performances of five different force fields currently used to describe Mg2+ in MD simulations are benchmarked, showing strengths and drawbacks. Moreover, the non-trivial electronic effects induced by Mg2+ on its ligands, such as charge transfer and polarization, are also characterized using 16 recurrent binding motifs. Overall, the study offers some guidelines on Mg2+ force fields for users and developers. Chapter 6 represents the final stop of the evolutionary journey. Here, an exquisite cryo-EM model of the ILS spliceosomal complex solved at 3.6 \uc5 resolution is used for a long-time scale MD study. This provides precious insights on the main proteins and snRNAs involved in the pre-mRNA splicing in eukaryotes as well as on the catalytic site. Unprecedentedly, the structural and dynamical properties of the spliceosome machinery are investigated at the atomistic level, with a particular emphasis on protein/RNA interplay through the characterization of their principal motions, among which the intron lariat/U2 snRNA helix unwinding

Fabrication and characterization of a polymeric nanofluidic device for DNA analysis

The growing needs for cheaper and faster sequencing of long biopolymers such as DNA and RNA have prompted the development of new technologies. Among the novel techniques for analyzing these biopolymers, an approach using nanochannel based fluidic devices is attractive because it is a label-free, amplification-free, single-molecule method that can be scaled for high-throughput analysis. Despite recent demonstrations of nanochannel based fluidic devices for analyzing physical properties of such biopolymers, most of the devices have been fabricated in inorganic materials such as silicon, silicon nitride and glass using expensive high end nanofabrication techniques such as focused ion beam and electron beam lithography. In order to use the nanochannel based fluidic devices for a variety of bioanalyses, it is imperative to develop a technology for low cost and high through fabrication of such devices and demonstrate the feasibility of the fabricated nanochannel based fluidic devices in obtaining information on biopolymers. We developed a low cost and high throughput method to build polymer-based nanofluidic devices with sub-100 nm nanochannels using direct imprinting into polymer substrates. Imprinting with the polymer stamps showed good replication fidelity for multiple replication processes, preventing damage of the expensive nanopatterned master and reducing undesirable deformation in the molded polymer substrate. This approach opened up a possibility to build cheap and disposable polymer nanofluidic devices for single molecule analysis. The ion transportation and DNA motion in nanofluidic systems were studied. Simulation and experiment results indicate that fast degeneration of the electric field at micro/nano interface plays a major role, in addition to the bulk flow in the microfluidic networks. Inlet structures and bypass microchannels were designed and built, the use of which has proven to enable enhancing the DNA capture rate by over 500 %. Attributed to the improved capture rate, the blockade current of DNA translocation though a nanochannel was also measured. We observed in the current versus time curves both current increase and decrease in the existence of a DNA molecule in the nanochannel, which we attributed to the ion channel blockage and electrical double layer formed around the DNA molecule, respectively

A spin-label ESR study of drug binding to DNA

A spin-labelled derivative of the drug Proflavine was prepared and its interactions with natural DNA in fibres were investigated using the technique of electron spin resonance (ESR). Computer simulations of the ESR spectra showed that spin-labelled proflavine adopts a preferred orientation in each of the different DNA conformations. In A- and C-form DNA the binding geometry is explained by an external interaction between the drug molecules and the phosphate groups of the double helix. In B-form DNA the drug molecules exhibited some form of rotational motion. Simulations indicated that this motion was directed about the helix axis, a conclusion which is consistent with an intercalative mode of binding. An examination of the B-form X-ray diffraction patterns confirmed that intercalation had occurred. A difference was observed in the binding of the drug to different species of DNA, suggesting that some form of site specific interaction is involved. The importance of drug concentration on the conformational properties of DNA was recognised. The binding of several Phenothiazine tranquilisers to DNA was also investigated. The solution ESR spectra of these (oxidised) drug molecules showed that the distribution of unpaired spin density over the heterocyclic ring depends upon the substituted groups present. However, in DNA fibres the different drug species adopted the same binding geometry. Although the ESR results were consistent with both intercalation and external binding, the large spread of drug orientations estimated from computer simulations suggests that external binding is more likely. DNA from the bacteriophage dW-14 was spin-labelled in order to determine the conformation of the putrescine groups attached to the thymine bases. The information obtained from these experiments suggests that the putrescine groups project into the major groove in B- and C-form DNA, whereas in the A-conformation they lie close to each other in the hollow interior of the helix

Basic Cell and Molecular Biology 5e: What We Know and How We Find Out

https://dc.uwm.edu/biosci_facbooks_bergtrom/1014/thumbnail.jp

A study of protein-DNA interactions using atomic force microscopy and DNA origami

The development and application of genome editing tools has accelerated in recent years. However, their widespread application, especially in the medical field, is delayed for various issues with the safety of the tools being one of the top concerns. Much of the detail of how proteins find their target sites and how they cleave at the sites remains unclear. Despite much progress in lab environments, where the tolerance for imprecise cutting is relatively high, in vivo treatment remains difficult. Most genome editing tools are derived from naturally occurring regulatory proteins. Better understanding of the mechanisms used by these natural proteins should facilitate the use of genome editing tools. In nature, gene regulation is usually sparked by a change in the cellular environment, such as viruses invading a bacterium. Restriction enzymes defend the host bacteria by recognising specific sites on the viral DNA and cutting invading viruses at those sites. We aim first to understand how these proteins translocate along the viral DNA molecules toward their recognition sites, and then to see how their accuracy of cleavage can be increased. Protein translocation along DNA molecules has been studied for more than 40 years. Atomic force microscopy in fluid mode allows direct observation of protein/DNA interactions. This application is about twenty years old but most of the images taken, lack the spatial and temporal resolution for quantitative studies of protein translocation dynamics. Here we achieve second-level and nanometre-scale tracking of the translocation of EcoRV, a Type IIP restriction enzyme, using fast-scan atomic force microscopy (AFM) and DNA origami techniques. We find that EcoRV tends to jump toward its recognition site from afar and then switch to slow sliding mode when it is within about 20 base pairs of its recognition site. Our methods demonstrate how BcgI, a type IIB restriction enzyme, brings together two recognition sites both in cis and in trans before cleavage, minimising mis-cleavage at a single recognition site. We show that the collision looping model is valid but not the sliding model. The two restriction enzymes were chosen as they represent different model systems of typical restriction enzymes. Our methods will be useful in studies of other types of restriction enzymes and other proteins or protein complexes that interact with DNA. We expect that these methods will see broader applications in studies of protein-DNA interactions. We also hope that our studies will contribute to the safe application of the genome-editing tools in medical contexts.Cambridge Trus

Annotated Cell and Molecular Biology 5e: What We Know and How We Found Out

https://dc.uwm.edu/biosci_facbooks_bergtrom/1013/thumbnail.jp

The Double Helix in Motion: New Insights into Sequence-specific, Functional DNA Dynamics Using NMR Spectroscopy

DNA is a highly flexible molecule that undergoes a variety of structural transitions in response to cellular cues. Sequence-directed variations in the canonical double helix structure that retain Watson-Crick base-pairing play important roles in DNA recognition, topology, and nucleosome positioning. Here, we use NMR relaxation methods to study sequence-directed dynamics occurring at picosecond to millisecond timescales in variable size DNA duplexes. Traditionally, atomic-level spin relaxation studies of DNA dynamics have been limited to short duplexes, in which sensitivity to biologically relevant nanosecond fluctuations is often inadequate. We introduce a method for preparing residue-specific 13C/15N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply it towards probing fast inter-helical bending motions induced by an adenine tract. Our results suggest the presence of elevated A-tract independent end-fraying and/or bending internal nanosecond motions, which evade detection in short constructs and that penetrate deep within the helix and gradually fade away towards its interior. By studying picosecond-nanosecond dynamics in short DNA dodecamers with variable length A-tracts, we discover that A-tracts are relatively rigid and can modulate the flexibility of their junctions in a length-dependent manner. We identify the presence of large-amplitude deoxyribose internal motions in CA/TG and CG steps placed in different sequences that likely represent rapid sugar repuckering. Moreover, by using NMR relaxation dispersion in concert with steered molecular dynamics simulations, we observe transient sequence-specific excursions away from Watson-Crick base-pairing at CA/TG and TA steps inside DNA dodecamers towards low-populated and short-lived A•T and G•C Hoogsteen base pairs. We show that their populations and lifetimes can be modulated by environmental factors like acidity, monovalent and divalent ions as well as intrinsic sequence and chemical modifications. The observation of Hoogsteen base pairs in duplexes specifically bound to transcription factors and in damaged sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base pairs as a means of expanding its structural complexity beyond Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.Ph.D.Chemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89648/1/nikolove_1.pd