1998 — 2007 |
Stargell, Laurie A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetic Analysis of Transcription Initiation @ Colorado State University-Fort Collins
DESCRIPTION: The long-range objective of this proposal is to understand the process of transcription initiation by RNA polymerase II (Pol II) in vivo. The focus is on the function of TATA-box binding protein (TBP), a highly conserved protein found in all eukaryotes. Binding of TBP to the TATA-box in promoters of genes transcribed by Pol II initiates assembly of the remainder of the transcription machinery. Due to this critical function, it is important to identify factors that interact with TBP and determine how these interactions regulate TBP activity. A combination of techniques, including classical and molecular yeast genetics, recombinant DNA technology, and protein biochemistry, will be used to accomplish the following. First, TBP functions involved in activated transcription by Pol II in vivo will be determined. Two distinct mechanistic classes of activation-defective TBP alleles have been described: defective for recruitment of TBP to the TATA-box; and defective after TBP arrives at the TATA-box. Interactions between these TBP mutants and key components of the transcription machinery will be examined. Also, unlinked suppressors of these mutations will be isolated, the genes cloned and studied. Second, the role of TFIIA in transcription will be investigated. Since the TBP-TFIIA interaction is essential for activation in vivo, factors capable of recognizing TFIIA or the TFIIA-TBP complex, will be identified and characterized. Additional functions of TFIIA will be defined by mutational analysis and subsequent suppression of the mutant phenotypes. Third, the mechanisms of transcription at TATA-lacking Pol II promoters will be defined in vivo. Not all Pol II promoters contain TATA-boxes, although TBP is still required. Using a TBP mutant specifically defective for TATA-less transcription, factors will be identified (by genetic and biochemical approaches), that play a unique role in transcription from TATA-less promoters. Fourth, genetic selections will be used to identify new alleles of TBP defective in other aspects of Pol II transcription, thereby uncovering novel TBP functions. In summary, the proposed experiments are intended to provide information on the function of TBP and TBP-interacting factors in eukaryotic gene regulation. The factors and mechanisms of transcription initiation are conserved from yeast to humans. Thus, these results will be directly relevant to higher eukaryotes, where changes in gene expression are critical aspects of cell growth, development, and the response to environmental stimuli.
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0.958 |
2009 — 2014 |
Stargell, Laurie |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Transcriptional Regulation During Oxidative Stress @ Colorado State University
Project Abstract: This project directly addresses the fundamental biological question as to how genes are turned on and off in response to an environmental stimulus. Yeast is used as the model system and the response to oxidative stress as the stimulus. The yeast system allows for the identification and characterization of important players in living cells (in vivo), followed by expansion and refinement of these studies at the mechanistic level (in vitro). This multidisciplinary approach involving genetic, molecular, and biochemical techniques provides research opportunities for students at various skill levels. In addition, the oxidative stress response is a critical physiological response that is extremely important to cell survival in an oxygen-rich environment. Indeed, oxidative stress is also highly relevant to the human condition because it plays fundamental roles in aging, neurodegenerative diseases, and cancer. With the goal of understanding transcriptional themes in response to oxidative stress, this project explores two main areas: the role of regulatory factors in the response to oxidative stress and the mechanism(s) by which a single activator can differentially regulate a wide array of genes. Taken together, these combined studies will advance the understanding of the transcriptional response to oxidative stress, and will add to the current knowledge of the ways in which genes are turned on and off in response to an environmental change.
In addition to the intellectual merit, the project will have broad impact in at least four ways. First, many aspects of these studies will provide suitable projects for undergraduate trainees, who may have time constraints as well as limited laboratory experience. Second, studies with higher technical and time demands will provide outstanding cross-training experiences for graduate students in a number of different disciplines (biochemistry, genetics, molecular biology). It is important to note that a majority of these techniques are versatile. Trainees could certainly utilize these techniques to investigate other critical genetic mechanisms (DNA repair, replication, recombination, RNA metabolism, epigentics, etc.) in their future careers. Third, to propagate the tradition of outreach, both undergraduate and graduate students will be involved in taking gene expression studies into elementary school classrooms. Fourth, results from this project will be published in broad based scientific journals, and presented at local and international meetings. Taken together, this project directly supports the mission of the NSF to advance discovery and understanding, while promoting teaching, training and learning.
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1 |
2010 — 2014 |
Stargell, Laurie A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 3 @ Colorado State University
Dynamic transitions in chromatin stmcture are key elements to normal pattems of transcriptional activation in vivo. This Program of Projects is designed to fill a void in our understanding of how histone chaperones, in conjunction with histone acetylti'ansferases (HATs), contribute to the regulation of chromatin structure. Our approaches are broad and multi-pronged, spanning from in vivo genetic and molecular studies, to in viti'o biochemical and sti'uctural investigations. These innovative studies are highly synergistic and are dependent on the services provided by three Cores Facilities. The three Research Projects will pursue three common hypotheses. The aims proposed in the Stargell Project specifically address tiie contributions of chaperones and HATs to gene expression programs in vivo. The Stargell Project will test tiie first hypothesis (histone mobilization by chaperones is linked to histone acetylation) by determining whetiier there are specific in vivo contributions to gene expression, chromatin organization and histone mobility that are distinct for different chaperones and HAT family members. To test the second hypothesis (histone chaperones interact with histone acetyltransferases), we will determine if chaperones are required for HAT activity in vivo (or vice versa), and whether physical interactions can be detected. For hypothesis 3 (histone chaperones fijnction beyond the mono-nucleosome), we will investigate the potential roles of histone chaperone in particular steps in transcription (PIC formation, elongation, etc.), and assay chaperone complexes purified from yeast cells for their in vitro capabilities. Our work will provide a detailed picture of the influence of histone chaperones and histone acetylation on the dynamic nature of chromatin during the activation of gene expression in vivo. Taken together, this Program of Projects will explore transitions in chromatin and the links between histone chaperones and HATs. The results generated will profoundly impact our understanding of how genome accessibility is regulated.
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0.958 |
2013 — 2017 |
Stargell, Laurie |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Poising of Rna Polymerase Ii: Features and Functions @ Colorado State University
Intellectual Merit. The initial discovery of the occupancy of RNA polymerase II (RNAPII) at certain genes prior to their transcriptional activation occurred over a quarter century ago in Drosophila. Due to recent advances in whole genome analyses, occupancy of RNAPII in an inactive state can be detected in organisms across the evolutionary spectrum and at diverse sets of genes. The goal of this project is to investigate post-recruitment mechanisms of gene regulation by focusing on poised RNAPII in yeast and the response to oxidative stress as the regulatory system. These studies take full advantage of the available approaches (genetics, molecular biology, biochemistry, cell biology, genomics), and are amenable to researchers at a variety of levels (graduate, undergraduate and elementary school students). Thus, the transcriptional response to oxidative stress will be investigated with a focus on the pre-bound and poised RNAPII complex by testing two overarching hypotheses: 1) Poised promoters share common features that are distinct from recruitment-regulated promoters; and 2) Poised promoters offer functional advantage(s) for the regulation of gene expression. Since post-recruitment regulation is widespread in humans and other organisms, these findings will significantly impact the field of gene expression with exceptional potential to alter our overall views. Thus, these studies are extremely timely and highly significant.
Broader Impacts. The project has broad impact in at least four ways. First, the studies offer suitable projects for undergraduate trainees, who may have time constraints as well as limited laboratory experience. Second, studies with higher technical and time demands provide outstanding cross-training experiences for graduate students in a number of different disciplines (genetics, genomics, cell and molecular biology). These techniques are highly versatile. Trainees will utilize these techniques in the future if they choose to investigate other critical genetic mechanisms (DNA repair, replication, recombination, RNA metabolism, epigenetics, etc.). Third, to propagate the tradition of outreach, undergraduate and graduate students will be involved in taking gene expression studies into elementary school classrooms. As part of a previously funded NSF project, fifth graders and their teachers helped to develop a novel outreach program called 'Biochemistry is Elementary'. This innovative program has eight separate 1 hour sessions involving hands-on activities designed to explore basic concepts of scientific reasoning involving biochemistry and yeast genetics. Due to the inexpensive and biologically safe reagents and the detailed workbooks created during the project, many scientists could offer something similar to their local community if they received training on the program. As such, this program will be expanded to include an interactive website. Finally, results from this project will be published in broad based scientific journals, and presented at local and international meetings. Taken together, this project directly supports the mission of the NSF to advance discovery and understanding, while promoting teaching, training and learning.
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1 |
2019 — 2022 |
Rappe, Anthony (co-PI) [⬀] Stargell, Laurie |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Application of Hydrogen Bond Enhanced Halogen Bond For Biomolecular Engineering @ Colorado State University
Halogens (fluorine, chlorine, bromine, and iodine) are elements found in 25% of drugs currently used to treat human disease. The role of halogens in providing drugs with their specificity against clinical targets is now recognized as coming from a chemical interaction called the halogen bond. The halogen bond is similar to the better-recognized hydrogen bond, which is responsible for holding the structures of DNA and proteins together. With this award, the Chemistry of Life Processes program of the Chemistry Division is funding Professor Laurie Stargell and Professor Anthony Rappe to determine how hydrogen bonds enhance the strength of halogen bonds, allowing this now stronger interaction to be engineered to design new proteins. The enhanced halogen bonds will be used to create proteins that interact with other proteins, leading to a new computer method to design drugs to treat human disease. In addition, the enhanced halogen bonds stabilize proteins that are otherwise not entirely stable, which has been associated with neurodegenerative diseases such as Alzheimer's disease. This project will provide training in structural and computational chemistry for graduate and undergraduate students, including those from underrepresented groups. Finally, an outreach project will help middle school and high school students learn how scientists use 'X-ray vision' to see the atoms in DNA and proteins.
The hydrogen bond (HB) is a well-characterized and prevalent interaction in biology. The halogen bond (XB) is increasingly being recognized as important in chemistry and biology, particularly in biomolecular engineering and in the design of inhibitors against biomolecular targets. Very recently, it was shown that an HB donor can significantly increase the XB potential of an adjacent halogen substituent through a variation called an HB-enhanced XB (or HBeXB, for short). This project will test the hypothesis that HBeXBs are strongly stabilizing interactions that can be exploited in biomolecular engineering and inhibitor design. In order to realize the potential of the HBeXB as a design tool, its structure-energy relationships must be characterized and incorporated into a molecular simulation algorithm. The objectives of this project are to determine the prevalence of HBeXBs in the chemical and protein structural databases; incorporate HBeXBs into an empirical force field for halogen bonds to accurately model this synergistic interaction; and engineer HBeXBs into coiled coil peptides and partially stable proteins to validate this new force field and as a proof of concept for bimolecular engineering.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
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