New insights into stop codon recognition by eRF1

In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket

Studies of high molecular weight systems by NMR spectroscopy

Proteins are essential components for all living cells. For better understanding of proteins and how they work, it is essential to know their 3D structure as well as how they interact with other molecules and their dynamics. Nuclear magnetic resonance (NMR) spectroscopy is a versatile technique to study biomolecules, most notably proteins, in solution. NMR provides structural information at atomic resolution and, compared to the other techniques with similar resolution, the measurements can be carried out under near-physiological conditions that are meaningful for the investigation of dynamics. A great number of unnatural amino acids can now be incorporated into proteins both in vivo and by cell-free protein synthesis at genetically encoded positions. Isotopically labelled unnatural amino acids present outstanding probes for site-specific studies of proteins by NMR. In this thesis, the unnatural amino acid 13C-O-tert-butyltyrosine (13C-Tby) was incorporated as an NMR probe to study the proteins IMP-1 and the E. coli single-stranded DNA binding protein (SSB). IMP-1 is a clinically important metallo-beta-lactamase which catalyzes the hydrolysis of almost all beta-lactam antibiotics. In order to develop inhibitors against this enzyme it is important to understand its structure and dynamics. Pseudocontact shifts (PCSs) provide long-range structural information on biological macromolecules. In Chapter 2, PCSs were measured to study the conformation of an active site loop of IMP-1 and its change upon binding of a ligand. This loop is important as it lines the substrate binding site and contributes to substrate specificity. The results suggest that the binding of an inhibitor induces two different conformations in the loop which are in slow equilibrium between each other. E. coli SSB is a homotetramer of molecular mass 76 kDa. It binds single-stranded DNA (ssDNA) with high affinity and little sequence specificity in two main binding modes, named (SSB)65 and (SSB)35. Chapter 3 describes studies of ssDNA-SSB complexes at high and low salt concentrations by solution NMR spectroscopy. Furthermore, experiments were conducted to determine the binding polarity of ssDNA on SSB. The results obtained are in broad agreement with the unusual binding polarity reported by the crystal structure of a complex between E. coli SSB and a poly-deoxycytidine oligomer. In addition to using the tert-butyl group of a Tby residue as an NMR probe, the present thesis introduced a novel chemical tag containing a trimethylsilyl (TMS) group. Chapter 4 demonstrates the ability of the tert-butyl and TMS groups to deliver site-specific information in high-molecular weight systems without any isotope labelling. The approach was illustrated by measuring intermolecular nuclear Overhauser effects (NOEs) in the 95 kDa complex between SSB and ssDNA

NMR studies of the archaeal exosome complex

This study is about structural studies of the exosome from Sulfolobus solfataricus. It includes dynamics analysis (by using Nuclear Magnetic Resonance) of the complex in absence and in presence of RNA. Additional biochemical experiments (degradation assays, fluorescence anisotropy) allowed further description of the interaction exosome-RNA

Novel 19F NMR sensors for the characterization of higher-order secondary structures of DNA and RNA

Both DNA and RNA can fold into a variety of non-canonical structures. Non-canonical structures, such as triplexes and G-quadruplexes, are an active research area due to their biological significance and therapeutic potential. As structural complexity and conformational transitions are essential for the diverse biological roles of nucleic acids, characterization of the dynamic nature of nucleic acid is vital to understand their functional properties. This thesis focuses on 19F NMR spectroscopy as a tool that can be used to investigate the conformational polymorphism of nucleic acids and the dynamic nature of nucleic acid-ligand interactions. The utility of 19F NMR is based on covalently incorporated fluorine labels that act sensitive reporters upon conformational transition. In this study, six novel fluorine-labelled building blocks were synthesized and incorporated into oligonucleotides applying standard solid-phase oligonucleotide synthesis. The building blocks were successfully used to investigate DNA and RNA triplexes, RNA invasion and bistable hairpin-G-quadruplex RNA structures. Melting of triplexes could be followed from well-distinguish 19F signals of the triplex, duplex and single strand species, and melting temperatures of the structures were obtained. The temperature dependent 19F NMR data of the bistable RNAs enabled to characterize melting processes, melting temperatures and thermodynamic parameters. In addition, ion induced changes at the hairpin-G-quadruplex equilibrium positions were successfully monitored. In general, the 19F NMR experiments provided new information on investigated structures and demonstrated that five of the building blocks can be considered suitable for further 19F NMR applications

Function and dynamics of aptamers: A case study on the malachite green aptamer

Aptamers are short single-stranded nucleic acids that can bind to their targets with high specificity and high affinity. To study aptamer function and dynamics, the malachite green aptamer was chosen as a model. Malachite green (MG) bleaching, in which an OH- attacks the central carbon (C1) of MG, was inhibited in the presence of the malachite green aptamer (MGA). The inhibition of MG bleaching by MGA could be reversed by an antisense oligonucleotide (AS) complementary to the MGA binding pocket. Computational cavity analysis of the NMR structure of the MGA-MG complex predicted that the OH- is sterically excluded from the C1 of MG. The prediction was confirmed experimentally using variants of the MGA with changes in the MG binding pocket. This work shows that molecular reactivity can be reversibly regulated by an aptamer-AS pair based on steric hindrance. In addition to demonstrate that aptamers could control molecular reactivity, aptamer dynamics was studied with a strategy combining molecular dynamics (MD) simulation and experimental verification. MD simulation predicted that the MG binding pocket of the MGA is largely pre-organized and that binding of MG involves reorganization of the pocket and a simultaneous twisting of the MGA terminal stems around the pocket. MD simulation also provided a 3D-structure model of unoccupied MGA that has not yet been obtained by biophysical measurements. These predictions were consistent with biochemical and biophysical measurements of the MGA-MG interaction including RNase I footprinting, melting curves, thermodynamic and kinetic constants measurement. This work shows that MD simulation can be used to extend our understanding of the dynamics of aptamer-target interaction which is not evident from static 3D-structures. To conclude, I have developed a novel concept to control molecular reactivity by an aptamer based on steric protection and a strategy to study the dynamics of aptamer-target interaction by combining MD simulation and experimental verification. The former has potential application in controlling metabolic reactions and protein modifications by small reactants and the latter may serve as a general approach to study the dynamics of aptamer-target interaction for new insights into mechanisms of aptamer-target recognition

Chemical Modification Methods for Protein Misfolding Studies

Protein misfolding is the basis of various human diseases, including Parkinson’s disease, Alzheimer’s disease and Type 2 diabetes. When a protein misfolds, it adopts the wrong three dimensional structures that are dysfunctional and sometime pathological. Little structural details are known about this misfolding phenomenon due to the lack of characterization tools. Our group previously demonstrated that a thioamide, a single atom substitution of the peptide bond, could serve as a minimalist fluorescence quencher. In the current study, we showed the development of protein semi-synthesis strategies for the incorporation of thioamides into full-length proteins for misfolding studies. We adopted the native chemical ligation (NCL) method between a C-terminal thioester fragment and an N-terminal Cys fragment. We first devised strategies for the synthesis of thioamide-containing peptide thioesters as NCL substrates, and demonstrated their applications in generating a thioamide/Trp-dually labeled α-synuclein (αS), which was subsequently used in a proof-of-concept misfolding study. To remove the constraint of a Cys at the ligation site, we explored traceless ligation methods that desulfurized Cys into Ala, or β- and γ- thiol analogs into native amino acids after ligation in the presence of thioamides. We further demonstrated that selective deselenization could be achieved in the presence of both Cys residues and thioamides, expanding the scope of thioamide incorporation through traceless ligation to proteins with native Cys. Finally, we showed that hemiselenide protected selenocysteines (Sec) can be incorporated onto the protein N-terminus through chemoenzymatic modification by aminoacyl transferase (AaT) as ligation handles. Further developments are underway in our laboratory to expand the AaT substrate scope for β- and γ- thiol amino acid analogs. In summary, we developed a set of methods that allowed the incorporation of thioamide probes into full-length protein, which enabled the application of this minimalist probe in protein misfolding studies

The Role of Base Modifications on Tyrosyl-tRNA Structure, Stability, and Function in Bacillus subtilis and Bacillus anthracis

tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA)