Peter B. Moore - US grants

The interactions which lead to sequence specific binding of proteins to RNAs will be studied using E. coli 5S ribosomal RNA and its cognate proteins as the principal model system. The objective is to understand what kinds of structures in ribosomal RNAs, and RNAs in general, are recognized by sequence specific binding proteins, and what kinds of interactions lead to recognition on the part of the protein. Proton NMR (500 MHz) is the primary experimental tool to be relied up. Preliminary data indicate that these complexes give rich, assignable spectra suggesting that substantial progress towards identifying the parts of the RNA and protein which interact should be possible. Data of this kind should not only help our understanding of RNA-protein interactions, but also resolve long standing controversies about 5S RNA structure. At the same time crystallization studies will be pursued in an effort to launch a high resolution three dimensional study of these molecules.

Experiments will be done to elucidate the structure of the ribosome from E. coli and to relate that structure to its function in protein synthesis. Neutron scattering methods will be used to determine the positions of the 21 proteins in the 30S subunit of the ribosome. The mapping will be extended to include the positions of tRNAs in the A site and P site of the ribosome and to measure the relative positions of the large and small subunit. Nuclear magnetic resonance will be used to characterize one or more binding interactions involving ribosomal proteins and rRNA. Special attention will be given to 5S RNA and its specific binding proteins. The object is to identify the kinds of residues involved, the types of interactions and the source of specificity in these interactions. As an adjunct to these experiments; the RNAs and proteins used in the RNA-protein investigations will be used for crystallization experiments in an effort to open the way to the elucidation of their structures at high resolution. Protein synthesis is a fundamental metabolic process performed in essentially the same manner both in man and bacteria. Studies proposed above should contribute to our understanding of the mechanism of this process in all organisms by revealing the basis of the specific RNA-protein interactions on which the process depends and the geometry of the enzymatically active complex. Knowledge of this kind forms the infra structure on which future medical advances depend. More directly, knowledge of the details of bacterial ribosomes may be clinically relevant because many commonly used antibiotics act by blocking ribosomal steps in protein synthesis in bacteria.

Lasers provide the chemist and physicist with the means of carrying out high resolution, high sensitivity experiments using most of the infrared spectrum. If laser instrumentation is combined into a central laser laboratory, the research of a group of top-notch investigators can be effectively enhanced. The Department of Chemistry at Yale University will use this award from the Chemical Instrumentation Program and the Atomic, Molecular, and Plasma Physics Program to help acquire modern laser instrumentation. The areas of research that will be enhanced by the acquisition include the following: 1) Structure and bonding in transient chemical species 2) Oxygen and other atmospheric gasses: characterization of specific optically excited levels, decay rates and mechanisms 3) Atomic and molecular Rydberg state spectroscopy 4) Structures of ionic clusters.

The objective of this project is to determine the three dimensional structure and physical properties of several commonly occurring RNA secondary structure motifs. Among the motifs to be examined are helices containing non-Watson Crick base pairs, and bulged bases, the loops of hairpin structures, and pseudoknots. High field NMR and x-ray crystallography will be the tools used. The structures in question are important components of all naturally occurring RNAs. RNAs serve essential roles as both substrates and catalysts in the gene expression pathway. The information gained should help us understand how these molecules perform their critical biological functions. The systems to be studied are: (1) the helix IV-V region of the 5S RNA from E. coli, which poses interesting questions about base mismatches and protein binding, (2) the pseudoknot structure at the 5' end of U2 RNA, an RNA essential for splicing reactions in eukaryotes, and (3) several purely synthetic RNAs that have simple loops sequences and secondary structure irregularities.

9317832 Brunger Computational approaches are important for structure determination by X-ray crystallography and solution NMR spectroscopy, studies of macromolecular structure/function relationships, protein folding, structure prediction, and drug design. The proposed workstation cluster will enable us to extend the limitations of these methods and to apply them to more difficult and important biological problems. The workstation cluster will also serve as a platform for testing advanced algorithms for the parallelization of our programs. The main computational tool for the proposed projects is the program X-PLOR (Brunger, 1992b). We routinely use X-PLOR for structure determination and refinement of X-ray and solution NMR structures, as well as for free energy perturbation calculations, structure prediction and certain aspects of structure-based drug design. It is proposed to acquire a workstation cluster which provides the most cost-effective and flexible solution to satisfy our needs. X-PLOR performs very efficiently on the proposed workstations. We intend to primarily use the cluster in a mode where multiple and independent jobs are run independently on the processors. Thus, we can readily make use of the proposed cluster without extensive software development.

The ribosome catalyzes messenger RNA-directed protein synthesis by a mechanism common to all organisms that remains obscure despite decades of study. It is important that we understand how ribosomes perform their function for many reasons: (1) protein synthesis is a critically important synthetic process in all organisms, (2) the mechanism may involve novel enzymology due to the intimate involvement of RNA in ribosome function, and (3) knowledge of the mechanism is likely to have clinical impact because bacterial ribosomes are the targets of many antibiotics. Since lack of high-resolution information about the conformation of the ribosome and the macromolecules that interact with it is seriously limiting the process of this field, the focus of the work planned for the next four years is the determination of the structure of the ribosome, and of some of its macromolecular ligands by X-ray crystallography. Three projects will be undertaken. First, the crystal structure of the large ribosomal subunit from Haloarcula marismortui will be solved. Crystals are available that diffract to better than 4 A, and a first heavy atom derivative is already be in hand. A the same time, biochemical studies of the factors that limit the quality of these crystals and of ribosome crystals generally will continue, as will crystallization trials of complexes of elongation factor G (EF-G) with ribosomes. Crystallization trials will also be run with 70S ribosomes from several bacterial species. Second, domains of 30S ribosomal subunits will be prepared by reconstitution, and experiments done to find out if they can be crystallized. The crystallization of complexes of EF-G and RNA oligonucleotides derived from the sarcin/ricin and thiostrepton regions will also be explored, as will the crystallization of complexes of ribosomal protein L25 and oligonucleotides derived from 5S rRNA. The structures of all new crystals obtained that are suitable for crystallographic analysis will be pursued. Third, the crustal structure of elongation factor G (EF-G) complexed with a GTP analogue will be solved.

The mechanism of ribosome-catalyzed, messenger RNA-directed protein synthesis is fundamentally the same in all organisms, and it is important that it be fully understood for many reasons. First, protein synthesis is a major metabolic activity in all organisms. Second, novel RNA-dependent enzymology may be involved. Third, because so many antibiotics target bacterial ribosomes, an understanding of the details of the ribosomal phase of translation may have significant clinical implications. Since lack of atomic resolution information about ribosome conformation has serious limited progress in this area of inquiry for many years. The focus of the work proposed here is the determination of the structure of the ribosome and the complexes it forms with antibiotics and the macromolecules with which it interacts by X-ray crystallography. Four projects will be undertaken all in collaboration with R.A. Steitz to a greater or lesser extent. First, the effort to solve crystal structure of the large ribosomal subunit from Haloarcula marismortui, which is already well underway, will be brought to a conclusion. The crystals available diffract past A resolution, and interpretable electron density maps of the structure can be computed today to 5 A resolution. Second, crystals will be prepared of domains of the small ribosomal subunit, in hopes of obtaining information about the conformation of that subunit at resolutions significantly higher than those accessible using the crystals of intact small subunits currently available. Third, a program will be instituted the objectives of which is the determination of the crystal structures of isolated proteins from the large ribosomal subunit of H. marismortui, or of other archael species, to facilitate the interpretation of the electron density maps of the H. maris mortui 50 ribosomal subunit that are becoming available. Fourth, crystals will be prepared of ribosomes from eukaryotic species, with the ribosomes form yeast being the first objective.

This Program supports the study of macromolecules, such as catalytic RNAs, and macromolecular assemblies like the ribosome and the plasma membrane. The RNAS, ribonucleoproteins, and enzymes responsible for gene expression, the proteins that regulate expression, and membrane proteins will be emphasized over the next five years. The primary techniques used to characterize these molecules will be single-crystal X- ray diffraction, where possible, and X-ray scattering and high-resolution cryoelectron microscopy when only solutions or partially ordered samples are available. This Program will also support studies of the motions that occur both within biological macromolecules and between components of macromolecular assemblies as they function. Motion can be inferred from confidential differences observed when time average structures are determined for a given macromolecule under different environmental conditions or in different states of ligation. Of special interest are the motions that occur: (1) during the catalytic cycles of RNA and DNA polymerases and other enzymes, (2) in the course of protein synthesis as the ribosome proceeds through its elongation cycle, and (3) during the insertion of intrinsic membrane proteins into lipid bilayers and the passage of secreted proteins through membranes. Theoretical investigations will also be undertaken of protein-membrane interactions, and work will continue on improving the computational procedures used to obtain structures both by S-ray crystallography and by NMR.

We request funding so that the console of the Varian Unity 500 MHz NMR spectrometer housed in the Yale Department of Molecular Biophysics and Biochemistry Bass Center can be replaced, and a cryoprobe purchased for that spectrometer. The Varian Inova console requested will replace a Unity console that was built ca. 1990, and was purchased by Yale, second hand, in 1995. Inova consoles have a more robust computer interface that requires fewer reboots, and that executes pulses, power level, phase changes and frequency changes more efficiently than Unity consoles. This upgrade should result in significantly improved implementations of pulse sequences. In addition, Inova consoles are compatible with Varian newest software, SPINCAD, which do3es not run on Unity or Unity plus systems. Thus this upgrade should ensure the usefulness of our 500 MHZ spectrometer for years to come. Last, but not least, replacement of the console in question will reduce the amount of instrument time lost for repair, which has become considerable because the Unity console is reaching the end of its useful life. The cryoprobe requested has recently been developed by Varian. Its major attraction is its high sensitivity, relative to room temperature probes. The sensitivity of the 500 MHz spectrometer equipped with a cryoprobe should be double the sensitivity observed with the best room temperature probe now available on the Yale 800 MHz spectrometer. There are several structural biology projects under consideration that demand this sensitivity because the molecules of interest aggregate at the concentrations required when standard probes are used.

DESCRIPTION (provided by applicant): Funds are requested so that two of the X-ray diffractometers used for macromolecular crystallography by investigators in the Yale Faculty of Arts and Sciences can be upgraded. The upgrade has two components: (1) replacement of a Rigaku RU300 rotating anode generator that currently supplies X-rays to both an R-Axis IV and a MAR 345 diffractometer, and (2) replacement of the associated X-ray optics. The RU300 generator in question, which was purchased in 1986, is increasingly prone to failure due to age, and even when operating properly, the X-ray beams it produces are not well matched to the crystallographic problems now being pursued, which involve smaller crystals than those usually studied in the 1980s, when the RU300 was purchased, and many of these crystals have large unit cell dimensions, which makes data collection even more challenging. The plan is to replace this generator with a Rigaku MicroMax-007 rotating anode generator. Not only should this generator be more reliable than the RU300 because it is newer, but because it is a microfocus generator, everything else being equal, its smaller, brighter beam should also enable investigators to collect diffraction data from almost all the crystals they are now studying much faster and more accurately than is now possible. In order to take full advantage of this MicroMax-007 generator the Osmic monochromators now mounted on the RU300 generator, which were built for generators that produce large beams, will have to be replaced with the VariMax optical components Rigaku recommends. Funds are requested for two such optical systems, so that both of the area detectors currently installed on the RU300 generator can be mounted on the new generator. It is anticipated that this upgrade will significantly increase the rate at which structures are determined at Yale.

The CORE laboratory provides facilities to all members of the Yale Center for Structural Biology for the[unreadable] investigation of macromolecular systems by X-ray crystallography and X-ray small-angle scattering, including[unreadable] both instruments for data collection, and computers for data reduction and analysis. Among the computers[unreadable] available are several molecular graphics systems, which are vital to the activities of all users. The staff of[unreadable] the CORE laboratory maintains and upgrades CSB instrumentation, and assists users with data collection[unreadable] and analysis. The CORE staff also maintains the CSB website, which is heavily used by structural biologist[unreadable] both inside and outside Yale. This invaluable resource provides its users with access to all the programs[unreadable] and data bases that are generally useful to structural biologists. The programs on the web site are upgraded[unreadable] as new releases appear, and documentation is provided for many of them to further assist users.