1985 — 2010 |
Kustu, Sydney G |
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. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Genetic Studies of Glutamine Synthetase in Bacteria @ University of California Davis
Synthesis of a number of bacterial proteins including glutamine synthetase, amino acid transport components and degradative enzymes, and nitrogenase is increased under nitrogen limiting conditions. In enteric bacteria increased synthesis appears to be mediated by products of nitrogen regulatory genes ntrA, ntrB, and ntrC, which activate transcription. Our major goals are to determine functions of the individual ntr products in regulating transcription and to identify sites to which they bind within the promoter-operator for glnA, the structural gene encoding glutamine synthetase. We will attempt to demonstrate regulatory effects of ntr products on glnA expression in vitro.
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1 |
1988 — 1999 |
Kustu, Sydney |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Regulation of Expression of the Nitrogen Fixation (Nif) Genes of K. Pneumoniae @ University of California-Berkeley
9405733 Kustu The NIFA protein (nifA gene product) is required to activate transcription of the nitrogen fixation (nif) operons in a wide variety of free-living and symbiotic bacteria belonging to the large gram-negative division proteobacteria. To activate, NIFA binds to enhancer-like sites approximately 100 bp upstream of nif promoters and allows RNA polymerase (alpha54-holoenzyme form) to denature the DNA strands around a transcriptional startsite -- that is, to isomerize from closed to open complexes. We succeeded in purifying the insoluble NIFA protein from Klebsiella pneumoniae as a fusion to the soluble maltose-binding protein (MBP) and in demonstrating its binding to enhancer-like sites in vitro for the first time. MBP-NIFA activated transcription by alpha54- holoenzyme in a purified system and could be shown to catalyze the isomerization of closed complexes between this polymerase and the nifH promoter to open complexes. Activation and open complex formation required a nucleoside triphosphate with a hydrolyzable, beta-gamma bond but we were unable to demonstrate hydrolysis directly because we could not remove contaminating hydrolytic activities from the preparation. We also purified an MBP fusion to just the central catalytic domain of NIFA, in the absence of its DNA-binding domain, and released the central domain in a soluble, active form by proteolytic cleavage of the fusion protein . The released central domain could activate transcription from solution, a property so far unique among enhancer-binding proteins, and had the expected ability to hydrolyze nucleotides. A purified renatured form of the NIFL protein, which is known to inhibit NIFA activity in vivo in response to the presence of molecular oxygen or combined nitrogen, inhibited transcriptional activation by both MBP-NIFA and the central domain of NIFA. Since NIFL did not inhibit nucleotide hydrolysis by the central domain, we postulate that it interferes with protein-protein contact between this domain of NIFA and alpha54-holoenzyme. Our major goals for the next grant period are: 1) to obtain NIFL preparations with better activity; 2) to determine whether NIFL inhibits NIFA activity by interacting with NIFA stoichiometrically or covalently modifying it; 3) to study protein-protein interactions between NIFL and NIFA and between NIFA and polymerase; and 4) to determine the mechanism by which NIFL senses molecular oxygen. The studies are of interest with respect to understanding the interaction of enhancerbinding proteins with the* target RNA polymerases and with regard to understanding the various forms of regulation that occur in response to molecular oxygen. We hope that they will eventually be of use to others in increasing the efficiency of biological nitrogen fixation. %%% Crop productivity is often limited by the availability of nitrogen in a fornm suitable for synthesis of proteins and other large molecules characteristie of living organisms. (certain bacteria, eithel alone or in partnership with plants, have the ability lo convert nitrogen gas from the atmosphere to ammonia, a nitrogen fertilizer, in a process called biological nitrogen fixation. We are studying the NIFA protein, a major regulator of biological nitrogen fixation. Characterizing NlFA will contnbute to understanding, how organisms decode palticu ar portions of their DNA under appropriate conditions and may help to improve the biological productivn of nitrogen felti1izer. A major goal for the next grant period is to understand how the function of the NIFA protein is poisoned by oxygen gas in the air. ***
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0.915 |
1989 |
Kustu, Sydney G |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Gordon Conference On Biological Regulatory Mechanisms @ Gordon Research Conferences
We request partial support for the 1989 Gordon Research Conference on Biological Regulatory Mechanisms. The purpose of the Conference is to promote exchange among a diverse group of scientists who are making outstanding contributions to research in this field. The conference is noted for its wide focus: it considers regulatory systems in a variety of prokaryotic and eukaryotic organisms, with emphasis on both conceptual and methodological advances. The 1989 Conference will include two new topics 1) Evolutionary considerations in the study of biological regulatory mechanisms, which will emphasize diversity to be found within the eukaryotic kingdom and within the archaebacterial kingdom of prokaryotes and 2) Regulation of plant gene expression, an area of intense study and current interest. Other session topics, which represent areas of rapid progress, include: Signal transduction: 1 millisecond to 10 seconds. The apparatus of cell division, Regulation of cell cycle progression, Developmental pattern formation, Regulation of gene expression, and Macromolecular assemblies. We hope that the program will attract conferees from university, government and industrial settings in the U.S. and abroad. The format will include poster sessions as well as the traditional lecture/discussion sessions to encourage direct participation by as many conferees as possible.
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0.906 |
1999 — 2003 |
Kustu, Sydney |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Regulation of Expression of the Nitrogen Fixation (Nif) Genes of K. Pneumoniae and Studies of the Glnk-Amtb Operon @ University of California-Berkeley
Nitrogen must be obtained from the environment by all living things in order to synthesize compounds like proteins and nucleic acids. The NifA protein is required to activate transcription of the nitrogen fixation operons in a wide variety of proteobacteria. In all of them NifA activity is controlled by molecular oxygen (02) but the mechanism differs with the organism. In members of the y-subgroup, NifA activity is inhibited by a second protein, NifL. NifL can also inhibit NifA activity in the presence of combined nitrogen. Thus relief of NifL inhibition requires the absence of both 02 and combined nitrogen, and how such effects are coordinated is an important regulatory problem. NifL carries a FAD cofactor associated with its N-terminal domain. Reduction of the cofactor with the powerful reductant dithionite relieves NifL inhibition of NifA activity in a purified transcription system in vitro. The working hypothesis is that an unidentified iron-containing protein may be the physiological reductant for the FAD cofactor of NifL in Klebsiella pneumoniae. There is strong evidence that the nitrogen regulatory protein GlnK, a protein allosteric effector of a sort recently identified in all three domains of life, is also required for relief of NifL inhibition. It is postulated that GlnK enables reduction of the FAD co-factor of NifL under anaerobic conditions only when combined nitrogen is also limiting, e.g. by raising the mid-point potential for reduction into the physiological range. To test this hypothesis a combination of genetic, molecular biological and biochemical methods is being used to determine: 1) whether GlnK makes reduction of the FAD co-factor of NifL easier in vitro and thereby results in relief of NifL inhibition and 2) whether GlnK interacts directly with NifL. Although there was no previous report of a growth defect associated with disruption of the amtB (ammonium-methylammonium transport B) gene in bacteria, which lies downstream of glnK and in an operon with it, amtB mutant strains of enteric bacteria grow poorly at low concentrations of NH4+ at pH values below 7. Growth studies with a variety of mutant strains in combination with studies of transport of the NH4+ analogue l4Cmethylammonium are most parsimoniously interpreted to indicate that AmtB proteins, which are found in all three domains of life, transport the uncharged species NH3 rather than the charged species NH4, and that they facilitate the rate of diffusion of this species rather than concentrating it in an energy-dependent manner. Both interpretations are at odds with previous views from a number of other laboratories. To test them further, aim 3) is to study the role of the AmtB protein of enteric bacteria in acquisition of NH3 at pH 7.0, which must be done in continuous culture; aim 4)is to determine whether AmtB mediates loss of NH3 to the medium when strains are grown on poor nitrogen sources that generate it--i.e. whether diffusion is bi-directional; and aim 5) is to determine whether apparent concentrative uptake of 14Cmethylammonium by S. cerevisiae is due to "acid trapping" in yeast vacuoles. These studies are of interest with respect to understanding the coordination of transcriptional regulatory responses to different environmental signals, that is, in understanding how environmental signals are coordinated to control decoding of particular portions of an organism's DNA. They also bear on the functions of the enteric GlnK protein, part of a sophisticated "two-tiered" regulatory system for sensing nitrogen availability, and on those of AmtB, which is essential for acquisition of ammonia at low concentrations.
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0.915 |