Peter Hans von Hippel - US grants

DESCRIPTION (provided by applicant): In this renewal application we describe the progress of our research over the last four years on studies of the molecular mechanisms and controls that regulate the functions of DNA replication and RNA transcription complexes. Our studies have used primarily biophysical methods, and have primarily involved the replication complex of bacteriophage T4 and the transcription complex of E. coli. However, these systems feature essentially the same molecular mechanisms for 'driving' and regulating these central life processes as those that characterize 'higher organisms', including humans. As a result these studies provide good model systems to examine how human replication and transcription proceed at the fundamental level, and provide insights into what goes wrong at these levels in various forms of cancer and genetic diseases that often seem to involve minor kinetic or structural changes in the properties of these 'macromolecular machines'. During the last reporting period we completed a number of studies on the above mechanistic questions, using reconstituted replication or transcription complexes that carry out their functions with essentiall the same rates, fidelities and processivities as the in vivo versions of the same complexes within real cells. In these studies we placed fluorescent base analogue probes at defined positions within the nucleic acid frameworks of the reconstituted complexes, and then used fluorescent and circular dichroism spectroscopy at wavelengths great than 300 nm (an optical range in which the rest of the protein and nucleic acid components of the complexes are transparent) to monitor biologically relevant conformational changes at the probe sites. By these means we obtained significant information about replication and transcription mechanisms under steady state or equilibrium conditions. During the next reporting period we will follow up on preliminary studies that have shown that various versions of these same optical probe approaches can be used in more complex optical set-ups to permit two-dimensional fluorescence spectroscopic (2DFS) and single molecule Fluorescence Resonance Energy Transfer (smFRET) and Fluorescence Linear Dichroism (smFLD) measurements that can follow the kinetics of reactions within these complexes in 'real time' and with msec to msec resolution. This now gives us the opportunity to obtain local structural and dynamic information on changes at defined and biologically-relevant base analogue and DNA backbone probe sites, as well as to map transition states within individual rate-limiting molecular steps in transcription and replication. We believe that by using these approaches we can reveal new aspects of mechanisms and regulatory control systems that were previously inaccessible to direct experimental measurement, and should provide new and valuable fundamental information to understand cell processes and differentiation and to learn what goes wrong at the molecular level in related disease states.

In this proposal we outline a detailed physico-chemical study of the structure and interactions of the various protein and nucleic acid components of the bacteriophage T4 DNA replication complex. Building on the results of Alberts and Nossal and their coworkers, who have defined this in vitro system, as well as on our own earlier studies, we will use chemical and photochemical cross-linking experiments to determine structural and topological relationships and to localize protein-protein and protein-nucleic acid contacts. Studies of binding equilibria between various components, and the dependence of these binding constants on salt concentration, pH and other environmental variables will be carried out to determine the nature and the free energy contributions to complex formation and stability of various types of functional group interactions. Rates of DNA synthesis and replication fork movement, as well as the processivity and fidelity of the synthesis catalyzed by various partial replication systems will be examined in order to attempt to understand, on a molecular basis, the role each protein plays in the coupled (leading and lagging strand) DNA elongation phase of the integrated replication process, and the protein-protein and protein-nucleic acid interaction mechanisms involved in the partial reactions of the overall system.

Equipment is requested for the large scale cultivation of cells, bacteria, and viruses for the production of a variety of proteins under study by a number of the research groups in the Institute of Molecular Biology. The groups listed here will constitute over 75 percent of the use of the equipment. The remaining time will be used by other members of the Institute, almost all of whom have NIH grants and work on related problems. Typically, the problems being investigated are regulatory proteins generally present at exceedingly low levels. To obtain adequate quantities for study of their physiological, biochemical, and physical properties, a facility is needed that is capable of producing large quantities of bacteria, viruses, and cellular organelles.

This Biological Facilities Center proposal requests funds for the purchase of ancillary protein sequencing equipment, peptide synthesis and purification equipment, and equipment for x-ray structure determination of native and engineered proteins. The equipment will be shared by the staff of the Institute of Molecular Biology, one of the leading centers for the study of macromolecular structure and function, and its subsidiary program in Cell Biology. The equipment will be used to identify and/or study proteins involved in the mechanism and control of DNA replication and transcription, the effect of changes in primary structure on stability and tertiary protein structure, the interaction of subunits within protein complexes and between membrane proteins and lipids, the export of proteins by eukaryotic cells, and the mechanism of signal transduction in bacterial chemotaxis. This instrumentation will facilitate the collaborative research that has resulted in important scientific contributions in areas such as the stability of proteins, protein-DNA interactions, DNA replication and DNA transcription.

Electron Paramagnetic Resonance (EPR) spectroscopy is a key technique for the study of molecules that contain an unpaired electron. EPR measurements on inorganic, organometallic and organic materials under photochemical, electrochemical and thermal stimulation lead to an understanding of chemical structure that is unattainable by any other technique. The acquisition of EPR instrumentation enhances the ability of chemists to carry out frontier research. The Department of Chemistry of the University of Oregon will use an award from the Chemistry Shared Instrumentation Program to help purchase a state-of the-art EPR spectrometer. The areas of study in chemistry that will be enhanced include: 1) Radical clocks, a new type of radical probe 2) Novel stable nitroxide free radicals for applications as spin labels for biophysical studies 3) EPR of cyclic dihydropyrene aromatics 4) New inorganic multicomponent oxides via sol-gel synthetic routes 5) EPR as a probe of surface defects 6) 19-electron complexes A unique feature of the proposal is the way the EPR facility is managed, providing access to the entire science community within the University including Chemistry, Physics, Biology and Geology Departments.

In this competing continuing research grant application we propose to extend our ongoing studies on the basic principles of protein- nucleic acid interactions that underlie the regulation of gene expression. During this reporting period we plan to focus on an analysis of the E. coli RNA polymerase transcription complex, and will use thermodynamic, kinetic and crosslinking methods to analyze the structure and function of the steady-state transcription elongation complex, the nature of pausing in this complex, and the relation of this pausing to "factor-independent" and rho-dependent transcription termination. In these studies we will isolate and study specifically paused and stable lambdal and T7 phage elongation complexes. We will also use these complexes to examine the binding of various regulatory proteins (including sigma factor, nusA protein, rho protein, etc.) to the core polymerase of the elongation complex. In addition we will continue our studies of the mechanisms of transcript release by rho protein at rho-dependent transcript termination sites. Physico-chemical studies of the mechanisms of rho protein oligomerization, of RNA and ATP binding to rho. and of the RNA-dependent activation of rho ATPase will also be continued. Finally, we will continue to develop general theoretical and experimental methods for the study of protein-nucleic acid interactions. As before, we will attempt both to examine specific physiologically-active protein complexes involved in gene expression, and to elucidate some of the general principles that underlie the function of all such systems.

Protein translocation and folding are facilitated in the cell by helper proteins known as molecular chaperones. The heat shock protein 70 family (hsp70) of molecular chaperones is central to these processes. The goal of this research proposal is to elucidate the functions of a newly discovered chaperone called Cer1p, found in the endoplasmic reticulum (ER). Cer1p has limited homology with known hsp70s and may have some overlapping functions with the ER hsp70, Kar2p. Cer1p, like Kar2p, is important for translocation, though only for those proteins being translocated posttranslationally. A combination of genetic, molecular biological, and biochemical approaches will be employed to investigate the role of Cer1p in translocation and its restriction to a subset of the import routes. By generating rapid onset mutations long term adaptation to loss of Cer1p can be eliminated, and the direct functions of Cer1p can more conclusively be revealed. Multiple strategies are proposed to generate rapid onset mutations of cer1. Several approaches will be used to investigate the molecular basis for Cer1p's restriction to the posttranslational import pathway. Biochemical fractionation of detergent extracts will be performed to identify potential interacting partners with Cer1p and to determine if these interactions are pathway specific. Whether Cer1p directly interacts with translocating chains will be determined by crosslinking assays using microsomes. Initial pulse chase experiments suggest that Cer1p plays a role in protein folding. Funds are requested to complete a series of in vivo folding experiments to assess the relative contributions of Cer1p, Kar2p, and the hsp40 homolog Scj1p in protein folding in the ER. In addition, biochemical experiments with purified components are proposed to directly compare the properities and regulation of Cer1p and the true ER hsp70, Kar2p. Together, these studies are designed to investigate how the activities of these related chaperones are controlled to function in the translation and folding pathway in the ER.

An Affinity sensors IAys Optical Biosensor will be purchased for studies in assembly and regulatory reactions in replication and transcription, the energetics of assembly of the bacterial chemotaxis signaling complex, the role of SNARE proteins in controlling the specificity of membrane fusion in yeast, assembly and structure of phage and transcription complexes, assembly and function of the RNA polymerase II transcription complexes and the role of chaperones in protein folding.

With this Major Instruments Proposal Applications we request funds to purchase a Beckman Optima XL-I Analytical Ultracentrifuge and an Affinity Sensors IAsys Optical Biosensor Apparatus, to be used to upgrade and complete our armamentarium of physical biochemical equipment for the characterization of the structure and interactions of macromolecular complexes of biological importance. Six major research groups within the Institute of Molecular Biology at the University of Oregon have described specific projects that will be greatly facilitated by these items of equipment. The laboratories and research projects that have been proposed specifically are listed as follows: Peter von Hippel laboratory (Assembly and Regulatory Reactions in Replication and Transcription); Frederick Dahlquist laboratory (The Energetics of Assembly of the Bacterial Chemotaxis Signaling Complex); Tom Stevens laboratory (The Role of SNARE Proteins in Controlling the Specificity of Membrane Fusion in Yeast); Carlos Bustamante laboratory (Assembly and Structure of Phage and Transcription Complexes); Diane Hawley laboratory (Assembly and Function of the RNA polymerase II Transcription Complexes); and Gregory Flynn laboratory (Hsp70 Chaperones and Their Role in Protein Folding). In addition, other members of the Institute of Molecular Biology will use the requested equipment as their research projects become amenable to biophysical characterization.

Project Summary -- Abstract In this renewal application we describe recent progress in our studies of the molecular mechanisms and controls that regulate DNA replication, and outline our plans for the next four years. With this application we (Professors Andrew Marcus and Peter von Hippel) propose to further formalize our on-going and increasingly close collaboration in these studies by applying for this grant renewal as `joint' Principal Investigators (PIs). In earlier work on this grant the von Hippel lab largely focused on solution studies of the replication complex of bacteriophage T4 and the transcription complex of E. coli. We note that these systems involve essentially the same molecular mechanisms for `driving' and regulating these central life processes as do those of `higher organisms', including humans. As a result these studies provide good model systems to examine how human DNA replication and RNA transcription proceed at the fundamental level, and provide insights into what goes wrong at these levels in various forms of cancer and genetic diseases that often seem to involve minor kinetic or structural changes in the properties or control of these `macromolecular machines'. During the last reporting period we completed a number of studies on the above mechanistic questions, using reconstituted DNA replication or RNA transcription complexes that carry out their functions with essentially the same rates, fidelities and processivities as the in vivo versions of the same complexes. We proceeded largely by placing fluorescent base analogue probes, or internal cyanine dye probes and FRET pairs, at defined positions within the nucleic acid frameworks of the reconstituted complexes, and then used fluorescent and circular dichroism spectroscopy at wavelengths great than 300 nm (an optical range in which the rest of the protein and nucleic acid components of the complexes are transparent) to monitor biologically relevant conformational changes at and near the probe sites. By these means we obtained significant information about replication and transcription mechanisms under steady state or equilibrium conditions, and then followed up with very successful initial spectroscopy studies that showed that various versions of these same optical probe approaches can be used in more complex arrangements to permit two-dimensional fluorescence spectroscopic (2DFS) and single molecule Fluorescence Resonance Energy Transfer (smFRET) and Fluorescence Linear Dichroism (smFLD) measurements that can follow the kinetics of reactions within these complexes in `real time' with µsec to msec resolution. As described in the present proposal, these approaches now permit us to obtain local structural and dynamic information on conformational changes that occur at defined and biologically- relevant base analogue and DNA backbone probe sites, as well as to map transition states of individual rate- limiting molecular steps within in vitro reconstituted models of relatively complete DNA replication complexes. Using these approaches we are beginning to reveal aspects of mechanisms and regulatory control systems that have previously been inaccessible to direct experimental measurement.