Channeling in purine biosynthesis : efforts to detect interactions between PurF and PurD and characterization of the FGAR-AT complex

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.Vita.Includes bibliographical references.Purine biosynthesis has been used as a paradigm for the study of metabolism of unstable molecules. Both phosphoribosylamine (PRA) and N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) have estimated half-lives in vivo of seconds. In order to avoid metabolite decomposition, one strategy cells could employ is channeling-the direct transfer of a metabolite between enzyme active sites without diffusion into the bulk media. While kinetic evidence for channeling of PRA has been reported between phosphoribosylpyrophosphate amidotransferase (PurF) and glycinamide ribonucleotide synthetase (PurD), no evidence for a PurF:PurD complex has been found. In an effort to detect this complex, stopped-flow fluorescence spectroscopy was used to detect changes in PurF fluorescence that may result from interaction with PurD. Critical to the success of these experiments was incorporation of tryptophan analogs (4-fluorotryptophan and 7-azatryptophan) into the proteins in order to increase signal specificity for PurF. No evidence for a PurF:PurD interaction was found under any of the conditions tested. The implication of this finding is discussed with regard to the PurF:PurD channeling model. Like all amidotransferase enzymes (ATs), channeling of NH3 between glutaminase and AT active sites has been implicated in the formylglycinamide ribonucleotide amidotransferase (FGAR-AT).(cont.) In B. subtilis, the FGAR-AT is composed of three proteins: PurS, PurQ, and small PurL. The first characterization of the B. subtilis FGAR-AT complex was carried out, and it was determined that a complex between the three proteins can only be isolated in the presence of Mg2+-ADP and glutamine. By analogy to the Salmonella FGAR-AT, ADP is believed to be acting as a structural cofactor, while formation of a PurQ-glutamine complex is essential for assembly of the FGAR-AT. Subsequent biophysical studies have indicated that the physiologically relevant form of the FGAR-AT complex contains 2 PurS, 1 PurQ, and 1 small PurL. Further studies on PurQ have identified residues important for catalysis and complex formation, while insight into the small PurL active site has been obtained by studies on the T. maritima enzyme. The FGAR-AT complex provides a new system in purine biosynthesis to study metabolite transfer among weakly interacting proteins.by Aaron A. Hoskins.Ph.D

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation

Ribonucleoprotein Purification and Characterization Using RNA Mango

The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro

Heterochromatic sequences in a Drosophila whole-genome shotgun assembly

BACKGROUND: Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly. RESULTS: WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm. CONCLUSIONS: Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes

Population Genomic Inferences from Sparse High-Throughput Sequencing of Two Populations of Drosophila melanogaster

Short-read sequencing techniques provide the opportunity to capture genome-wide sequence data in a single experiment. A current challenge is to identify questions that shallow-depth genomic data can address successfully and to develop corresponding analytical methods that are statistically sound. Here, we apply the Roche/454 platform to survey natural variation in strains of Drosophila melanogaster from an African (n = 3) and a North American (n = 6) population. Reads were aligned to the reference D. melanogaster genomic assembly, single nucleotide polymorphisms were identified, and nucleotide variation was quantified genome wide. Simulations and empirical results suggest that nucleotide diversity can be accurately estimated from sparse data with as little as 0.2× coverage per line. The unbiased genomic sampling provided by random short-read sequencing also allows insight into distributions of transposable elements and copy number polymorphisms found within populations and demonstrates that short-read sequencing methods provide an efficient means to quantify variation in genome organization and content. Continued development of methods for statistical inference of shallow-depth genome-wide sequencing data will allow such sparse, partial data sets to become the norm in the emerging field of population genomics

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

On the issue of transparency and reproducibility in nanomedicine.

Following our call to join in the discussion over the suitability of implementing a reporting checklist for bio-nano papers, the community responds

Best Longitudinal Adjustment of Satellite Trajectories for the Observation of Forest Fires (Blastoff): A Stochastic Programming Approach to Satellite System Design

Forest fires cause a significant amount of damage and destruction each year. Optimally dispatching resources reduces the amount of damage a forest fire can cause. Models predict the fire spread to provide the data required to optimally dispatch resources. However, the models are only as accurate as the data used to build them. Satellites are one valuable tool in the collection of data for the forest fire models. Satellites provide data on the types of vegetation, the wind speed and direction, the soil moisture content, etc. The current operating paradigm is to passively collect data when possible. However, images from directly overhead provide better resolution and are easier to process. Maneuvering a constellation of satellites to fly directly over the forest fire provides higher quality data than is achieved with the current operating paradigm. Before launch, the location of the forest fire is unknown. Therefore, it is impossible to optimize the initial orbits for the satellites. Instead, the expected cost of maneuvering to observe the forest fire determines the optimal initial orbits. A two-stage stochastic programming approach is well suited for this class of problem where initial decisions are made with an uncertain future and then subsequent decisions are made once a scenario is realized. A repeat ground track orbit provides a non-maneuvering, natural solution providing a daily flyover of the forest fire. However, additional maneuvers provide a second daily flyover of the forest fire. The additional maneuvering comes at a significant cost in terms of additional fuel, but provides more data collection opportunities. After data are collected, ground stations receive the data for processing. Optimally selecting the ground station locations reduce the number of built ground stations and reduces the data fusion issues. However, the location of the forest fire alters the optimal ground station sites. A two-stage stochastic programming approach optimizes the selection of ground stations to maximize the expected amount of data downloaded from a satellite. The approaches of selecting initial orbits and ground station locations including uncertainty will provide a robust system to reduce the amount of damage caused by forest fires

Single molecule approaches for studying spliceosome assembly and catalysis

Single molecule assays of splicing and spliceosome assembly can provide unique insights into pre-mRNA processing that complement other technologies. Key to these experiments is the fabrication of fluorescent molecules (pre-mRNAs and spliceosome components) and passivated glass slides for each experiment. Here we describe how to produce fluorescent RNAs by splinted RNA ligation and fluorescent spliceosome subunits by SNAP-tagging proteins in cell lysate. We then depict how to passivate glass slides with polyethylene glycol for use on an inverted microscope with objective-based total internal reflection fluorescence (TIRF) optics. Finally, we describe how to tether the pre-mRNA onto the passivated slide surface and introduce the SNAP-tagged cell lysate for analysis of spliceosome assembly by single molecule fluorescence