Nils G Walter
University Of Michigan At Ann Arbor
Project start date: 2012-02-01
Project end date: 2015-11-30
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Nils G Walter
U-TURN OF THE HEPATITIS DELTA VIRUS RIBOZYME
Nils G Walter, Associate Professor
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 3R01GM062357-08S1 from National Institute Of General Medical Sciences
Abstract: The ultimate goal of this proposal is to understand catalysis by the HDV ribozyme. Ribozymes are ideal model systems for the vast number of non-protein coding RNAs found in all kingdoms of life. They have high biological and biotechnological relevance in their own right for their ability to process and regulate genetic information. Yet, two decades after their discovery, our understanding of ribozyme catalysis still pales compared to that of protein catalysis. A particularly intriguing and enigmatic example is the HDV ribozyme. It is the only ribozyme found in a human pathogen, the hepatitis delta virus (HDV), and has recently been discovered to also reside in the human genome. Multiple crystal structures exist that, together with detailed enzymatic studies, suggest a direct catalytic role of a specific nucleotide of the RNA chain, C75, as either general base or acid. During the previous funding cycle we discovered and characterized a conformational change that accompanies catalysis in solution and, as supported by subsequent crystallographic and molecular dynamics studies, appears to drive the catalyzed cleavage reaction forward. Most recently we have discovered that a ubiquitous U-turn motif is at the heart of this structural switch and exposes the cleavage site to the catalytic C75. This discovery links numerous, previously unconnected observations on the HDV ribozyme and offers the opportunity to fully characterize the complex relationship between RNA sequence, conformational change, folding free energy, and reaction chemistry, a first for any ribozyme. To take advantage of this opportunity and validate our methodology, we will employ an integrated set of biophysical and biochemical tools and will pursue the specific aims detailed below. Since the U-turn is one of the most common RNA structural motifs, our results promise to also have broad impact on our understanding of structural dynamics and function of the hammerhead ribozyme, tRNAs, HIV-1 genomic RNA, and GNRA tetraloops of large structured RNAs. In Specific Aim 1, we will experimentally measure the structural dynamics around the U-turn of the HDV ribozyme cleavage site by introducing (1) fluorophore pairs for single-molecule fluorescence resonance energy transfer (FRET) measurements of global conformational dynamics at the timescale of milliseconds and beyond; and (2) 13C,15N-labeled nucleotides as site-specific NMR probes to analyze local as well as global structural dynamics at the pico- to millisecond timescale. In Aim 2, we will solve the structure ensemble of the HDV ribozyme at atomic resolution and map its free energy landscape with advanced molecular dynamics simulations that will be guided by our experimental results from Aim1. In Aim 3, we will derive a full QM/MM of HDV ribozyme catalysis based on the structural dynamics map available from Aim 1 and 2. Finally in Aim 4, we will rigorously test our model of catalysis by introducing specific chemical modifications into the ribozyme´s U-turn, with the potential to improve catalytic efficiency of the naturally evolved HDV ribozyme wild-type
Keywords: AIDS Virus; Acids; Address; Anticodon; Biochemical; Biological; Biological Models; Catalysis; Catalytic RNA; Chemicals; Chemistry; Code; Coding System; Codon; Codon Nucleotides; Complex; Computer Simulation; Computerized Models; DNA Sequence Rearrangement; Delta Agent; Delta Virus; Dependence; E coli; Escherichia coli; FRET; Fluorescence; Fluorescence Resonance Energy Transfer; Free Energy; Funding; Gene Products, RNA; Genetic; Genome, Human; Genomics; Goals; HIV-1; HIV-I; HIV1; Heart; Hepatitis D Virus; Hepatitis Delta Virus; Human; Human Genome; Human immunodeficiency virus 1; Human, General; Immunodeficiency Virus Type 1, Human; In Vitro; Investigators; Label; Life; Link; Man (Taxonomy); Man, Modern; Maps; Mathematical Model Simulation; Mathematical Models and Simulations; Measurement; Measures; Method LOINC Axis 6; Methodology; Model System; Modeling; Models, Biologic; Models, Computer; Modification; Molecular Configuration; Molecular Conformation; Molecular Dynamics Simulation; Molecular Stereochemistry; Msec; Nucleotides; Process; Programs (PT); Programs [Publication Type]; Proteins; Quantum Mechanics; RNA; RNA Sequences; RNA, Non-Polyadenylated; Reaction; Rearrangement; Research Personnel; Researchers; Resolution; Ribonucleic Acid; Ribozymes; Role; Sampling; Science of Chemistry; Sequences, RNA; Simulation, Computer based; Site; Solutions; Structure; Testing; Validation; Variant; Variation; base; computational modeling; computational models; computational simulation; computational tools; computer based models; computer based prediction; computerized modeling; computerized simulation; computerized tools; conformation; conformational state; fluorophore; gene product; hammerhead ribozyme; human T cell leukemia virus III; human T lymphotropic virus III; improved; in silico; millisecond; molecular dynamics; molecular mechanics; movie; mutant; pathogen; predictive modeling; programs; single-molecule FRET; single-molecule fluorescence resonance energy transfer; smFRET; social role; tool; virtual simulation
Project start date: 2001-01-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PA-01-049
3R01GM062357-08S1 (2010): $53784
5R01GM062357-09 (2010): $323774
5R01GM062357-08 (2009): $268098
5R01GM062357-07 (2008): $268797
2R01GM062357-06A2 (2007): $269456
TREKKING WITH THE RIBOGNOME: SINGLE MOLECULE MICROSCOPY OF INTRACELLULAR MIRNPS
Nils G Walter, Associate Professor
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 5R01GM081025-04 from National Institute Of General Medical Sciences
Abstract: Currently, there are no suitable microscopy tools available that would allow researchers to follow the vast number of newly discovered, diverse non-protein coding (nc)RNAs around the cell as they fulfill their numerous biological functions, let alone at the single molecule level. The proposed project will fundamentally overcome this limitation by developing a novel probe concept optimized for detecting single small ncRNA molecules inside living cells. It is expected that our "molecular Christmas tree" probe technology will subsequently be transferable to other biopolymers. When developing a new cell microscopy technique, real-world field testing on a biological system for the purpose of determining performance parameters is essential. Among recently discovered ncRNAs are those associated with the new gene regulatory paradigm of RNA interference (RNAi), where in one pathway micro-RNAs (miRNAs) act to repress endogenous genes in all multicellular eukaryotes, including humans. A founding class member is let-7, or lethal-7, which is an evolutionarily conserved miRNA from C. elegans to humans. It has been found to regulate expression of disease-related transcriptional effectors, among them the High Mobility Group AT-hook 2 (HMGA2) protein involved in transcriptional regulation and associated with various cancers as well as diet-induced obesity. Expression of HMGA2 mRNA is controlled by an unusual seven let-7a-1 binding sites. As a proof-of-principle for our intracellular probe technology we will detect the assembly of let-7 miRNA, HMGA2 mRNA and RNAi proteins into single active micro-RNA-protein (miRNP) complexes, by pursuing the following milestones in collaboration with the groups of Sunney Xie (Harvard U.) and David Bartel (Whitehead Institute/MIT) (1) We will design, synthesize and test single molecule detection in cultured cells on a fully controllable sample. The necessary signal-to-noise threshold (amplification level) for intracellular detection of single assembled miRNP complexes is reached once a sufficient number of labeled let-7a probes are loaded onto a single target and slowly diffuse together in a complex. (2) We will test the developed probe technology on the real-world Let-7a/HMGA2 mRNA probe/target system and uniquely address numerous outstanding questions concerning the cell biology of miRNAs. (3) We will define the scope and limitations of our "molecular Christmas tree" probe technology. That is, in parallel to Aims 1 and 2 we will ask Is multiplexing (i.e., the detection of multiple targets in parallel) possible? Can multiple tandem repeat sequences in a DNA target be detected? Can protein assembly, particularly that occurring during protein misfolding diseases such as Alzheimer´s and prion diseases, be detected by our novel "molecular Christmas tree" probe concept? What are the lower limits for DNA repeat sequence and protein polymerization detection and can these limits be further pushed? Can multiple different biopolymers be detected in parallel and how does this affect detection limits?
Keywords: 3` Untranslated Regions; 3`UTR; Address; Affect; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer`s; Alzheimer`s Disease; Alzheimers Dementia; Alzheimers disease; Amino Acid Sequence; Assay; Benchmarking; Best Practice Analysis; Binding; Binding (Molecular Function); Binding Sites; Bioassay; Biologic Assays; Biological; Biological Assay; Biological Function; Biological Process; Biology; Biopolymers; Blastocytes; Blastomeres; C elegans; C.elegans; Caenorhabditis elegans; Cancers; Cell Communication and Signaling; Cell Line; Cell Lines, Strains; Cell Signaling; CellLine; Cells; Cellular biology; Code; Coding System; Collaborations; Color; Combining Site; Complex; Cultured Cells; Cytosol; DNA; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Deoxyribonucleic Acid; Detection; Diet; Diffuse; Disease; Disorder; Dissociation; Embryonic Cell; Equilibrium; Eukaryota; Eukaryote; Functional RNA; Genes; Genes, Regulator; Goals; HMG I-C Protein; HMGA2 Protein; HMGI-C; HMGI-C Protein; High Mobility Group AT-Hook 2; High Mobility Group Protein HMGIC Breakpoint Associated with Benign Lipoma; High Mobility Group Protein Isoform I-C; High-Mobility Group (Nonhistone Chromosomal) Protein Isoform I-C; High-Mobility Group Protein HMGI-C; Human; Human, General; Institutes; Intracellular Communication and Signaling; Investigators; Label; Life; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Messenger RNA; Methods and Techniques; Methods, Other; Micro RNA; MicroRNAs; Microscopy; Molecular; Molecular Biology, Protein Sequencing; Molecular Interaction; Noise; Non-Coding; Non-Coding RNA; Obesity; Pathway interactions; Peptide Sequence Determination; Performance; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; PrP Proteins; Primary Senile Degenerative Dementia; Prion Disease Pathway; Prion Diseases; Prion Protein Diseases; Prion Proteins; Prion-Induced Disorder; Prions; Process; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Protein Sequencing; Protein Structure, Primary; Proteins; Quelling; RNA Interference; RNA Silencing; RNA Silencings; RNA, Messenger; RNAi; Reactive Site; Regulator Genes; Research Personnel; Researchers; Resolution; Sampling; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Sequence-Specific Posttranscriptional Gene Silencing; Signal Transduction; Signal Transduction Systems; Signaling; Site; Spongiform Encephalopathies, Transmissible; Structure of blastomere; System; System, LOINC Axis 4; Tandem Repeat Sequences; Tandem Repeats; Techniques; Technology; Testing; Transcription Regulation; Transcriptional Control; Transcriptional Regulation; Transcriptional Regulatory Elements; Transmissible Dementias; Trees; aberrant protein folding; abnormal protein folding; adiposity; balance; balance function; base; biological signal transduction; biological systems; blastomere structure; cell biology; corpulence; corpulency; corpulentia; cultured cell line; dementia of the Alzheimer type; design; designing; disease/disorder; eukaryotida; fluorophore; gene product; mRNA; malignancy; member; miRNA; neoplasm/cancer; novel; obese; obese people; obese person; obese population; pathologic protein folding; pathway; polymerization; primary degenerative dementia; programs; protein complex; protein mis-folding; protein misfolding; protein sequence; regulatory gene; senile dementia of the Alzheimer type; single molecule; spongiform degeneration; spongiform encephalopathy; tool; trans acting element
Project start date: 2007-08-01
Project end date: 2011-07-31
Budget start date: 1-AUG-2010
Budget end date: 31-JUL-2011
PFA/PA: PAR-06-288
5R01GM081025-04 (2010): $254611
5R01GM081025-03 (2009): $257681
5R01GM081025-02 (2008): $245413
1R01GM081025-01 (2007): $240397
FOLDING AND FUNCTION OF HAMMERHEAD AND DELTA RIBOZYMES
Nils G Walter, Assistant Professor
University Of Michigan At Ann Arbor 3003 South State Street, Room 1040 Ann Arbor, Mi 481091274
Grant 5R01GM062357-05 from National Institute Of General Medical Sciences IRG: BIO
Abstract: adapted from applicant s ) Structural dynamics is key to the function of biological macromolecules. Catalytic RNA s, or ribozymes, serve as excellent model systems to study the function of nucleic acids as an essential class of biomolecules. In this project, the hammerhead and delta ribozymes will be studied by a unique array of biochemical and biophysical approaches to answer three fundamental questions First, how does the RNA strand fold into a three-dimensional structure? Second, how does this structure result in the acquisition of catalytic activity? Third, what role does structural dynamic play in folding and catalysis? Both the hammerhead and the delta ribozyme are well suited to such experiments, since both are small enough for comprehensive studies, yet complex enough that significant conformational changes occur during folding and catalysis. Both model systems catalyze the same chemical reaction, yet are structurally very distinct, suggesting that nature has found two radically different solutions for a common catalytic function. Specific Aims for both systems are (1) Map the folding pathway of the ribozyme-substrate complex structurally, kinetically, and thermodynamically; (2) Separate the roles of metal ions in folding and catalysis; (3) Identify and eliminate misfolding pathways. The importance of this work includes advancement of our understanding of the dynamics of RNA structure and how it leads to catalytic activity, and applying this understanding to improving health, through optimizing ribozymes for gene therapy and as biosensors.
Keywords: chemical structure function, conformation, nucleic acid structure, ribozyme, catalyst, chemical kinetics, thermodynamics
Project start date: 2001-01-01
Project end date: 2006-12-31
5R01GM062357-05 (2005): $221970
Sponsored Links Excellgen http://Excellgen.com
5R01GM062357-04 (2004): $275137
3R01GM062357-03S1 (2003): $17679
3R01GM062357-03S2 (2003): $25184
5R01GM062357-03 (2003): $221970
1R01GM062357-01 (2001): $222338
Nils G Walter
University Of Michigan At Ann Arbor
Project start date: 2001-01-01
Project end date: 2015-06-30
PROBING FAST FOLDING INTERMEDIATES OF THE HDV RIBOZYME
Nils G Walter, Assistant Professor
Illinois Institute Of Technology 3300 S Federal St Chicago, Il 606163793
Grant 2P41RR008630-090029 from National Center For Research Resources IRG: ZRG1
Keywords: X ray, biomedical resource, biophysics, hepatitis D virus, molecular dynamics, ribozyme, clinical research
Project start date: 2004-04-09
Project end date: 2005-03-31