Robert A. Zimmermann - US grants

This project entails the use of photochemical crosslinking techniques to investigate the structure of (a) the peptidyl-and aminoacyl-tRNA binding sites on E. coli ribosomes and (b) the binding sites for ribosomal proteins on E. coli ribosomal RNA. Specifically developed aryl azide reagents are attached non-photochemically to available thio or amino groups in selected tRNA molecules and ribosomal proteins. The modified components are then allowed to form specific complexes with the 70S ribosome or ribosomal RNA, as appropriate. Irradiation results in the photochemical decomposition of the azides to highly reactive nitrenes which can insert into neighboring molecules of the complex. In a number of cases, specific aminoacyl-tRNA molecules have been directly crosslinked to the RNA component of the 30S ribosomal subunit by irradiation with near ultraviolet light. The sites of insertion in ribosomal RNA will be determined by nucleic acid sequencing methods. Crosslinks to proteins will be investigated by chromatography and by one- and two-dimensional gel electrophoresis. The results of these experiments should aid in defining the spatial arrangement of constituents within functional macromolecular aggregates such as ribosomes.

This project will utilize photoaffinity labeling techniques to characterize the topography of tRNA binding sites on the E. coli ribosome, and in vitro mutagenesis to study structure-function relationships in ribosomal proteins and rRNA. (1) Photoreactive nucleosides will be introduced into tRNA molecules by chemical and enzymatic technique or by in vitro transcription. New reconstruction methods will allow us to place the modified bases at any desired position. The tRNA derivatives will be cross-linked tot he ribosome and the labeled components will be identified. Particular emphasis will be placed on determining the orientation of tRNA in the A,P and E sites, the identity of cross-linked residues in both rRNA and ribosomal proteins, and the nature of any changes that occur in the relative positions of tRNA and ribosome during peptidyl transfer and translocation. (2) New photolabile nucleosides will be prepared and their photochemistry assessed. In addition, suitably protected forms of photoreactive nucleoside monomers will be prepared for use in the automated, solid- phase synthesis of oligodeoxyribonucleotides. These synthons should be useful not only for the preparation of photoreactive tRNA derivatives, but also for antisense, catalytic or other RNAs where the proteins will be studied with the aid of site-directed mutagenesis in conjunction wit biochemical analysis. In particular, the structural features of protein S8 that are required for its specific interactions with 16S rRNA and spc operon mRNA will be further defined. Moreover, the role of L27 in 50S- subunit function will be investigated using a genetic system in which the activity of the ribosomes is entirely dependent upon the assembly of mutant protein into 50S subunits. As L27 is a target for a number of macrolide antibiotics, this work may shed new light on the structural aspects of antibiotic resistance. (4) Site-directed alterations will be introduced into several segments of the genes for 16S and 23S rRNAs using in vitro mutagenesis. The subjects of investigation include three lethal point mutations and their intragenic suppressor, all of which occur within a highly conserved sequence at the 3' end of 16S rRNA. The effects of mutations at two sites within the 23S rRNA will also be examined, the conserved G1945 region adjoining the 3' end of the P-site tRNA and the sarcin stem-loop which may mediate the influence of elongation factors upon 50S subunit structure.

This project will focus upon the structure, function and genetics of protein and RNA components of the prokaryotic ribosome. (1) The structural features within E. coli 16S and 23S rRNAs that account for their specific interaction with r- proteins S8 and L1 during ribosome assembly will be probed by site-directed mutagenesis and protein-rRNA cross-linking. In addition, the minimum rRNA segment required for protein binding will be determined to facilitate their detailed physical characterization in the future. (2) Over 40 mutants of E. coli r-protein S8 have been isolated on the basis of their inability to translationally regulate spc operon expression. The metabolic and physically stability of these proteins will be examined, and their RNA-binding properties evaluated. 2D NMR analysis of wild-type and mutants S8 will be used to define the structure of S8, and its RNA-binding domain, at high resolution. (3) Operons encoding components of the translation and transcription apparatus in several thermoacidophilic archaebacteria will be isolated and sequenced. Various approaches will be employed to gain insights into how the expression of these transcription units is controlled. The results will provide a better understanding of the evolution of gene regulation, as well as of gene organization, in this unusual group of organisms.

9818051
The expression of genetic information in all cells depends on specific, noncovalent interactions between protein and RNA molecules. Nowhere is the importance of such interactions better illustrated than in ribosomes, the complex ribonucleoprotein particles that carry out the biosynthesis of cellular proteins according to genetic information encoded in messenger RNA. Ribosomal particles are composed of roughly 55 different proteins, two large RNA molecules, and one small RNA molecule which assemble into a compact, functional structure owing to an intricate network of noncovalent protein-RNA interactions. Specific associations between ribosomal proteins and ribosomal RNAs are essential both for the assembly and for the structural integrity of the ribosome, and underlie the functional activity of this particle in protein synthesis. In the bacterium Escherichia coli, many of the proteins that associate with ribosomal RNA also regulate the expression of the ribosomal protein genes through specific interactions with the corresponding messenger RNAs. These complexes provide a rich source of material for investigating the molecular bases of protein-RNA interaction. The present research is directed toward elucidating the specific structural features that mediate the association of small-subunit protein S8 with its binding site in 16S ribosomal RNA and of large-subunit protein L1 with its binding site in 23S ribosomal RNA. Each of the protein binding sites has been narrowed down to about 30 nucleotides and that for protein S8 has been solved by NMR. Further insight into the nature of the S8- and L1-RNA interactions will be gained through site-directed mutagenesis, protein-RNA cross-linking, functional group analysis, SELEX, NMR and X-ray crystallography. Precise information about the structure of ribosomal protein-rRNA complexes is important not only for understanding the way in which several dozen unique ribosomal components spontaneously assemble into functional ribonucleoprotein particles, but also for comprehending the principles that underlie the many different protein-RNA interactions that play crucial roles in the expression of genetic information via the synthesis of DNA, RNA and protein molecules.

The expression of the genetic information stored in the DNA culminates in the synthesis of the proteins that define the properties of individual cells. Remarkably, the mechanism of this process is quite similar in all organisms, from bacteria to humans. Protein synthesis is carried out by compact globular particles known as ribosomes which consist of a large number of protein and RNA molecules arranged in an intricate and highly specific manner. The ribosomal particles guide the assembly of many other cellular components on its surface in a way that permits the efficient linkage of amino acids into protein chains according to the genetic plans encoded in the DNA. Ribosome function in protein synthesis depends critically on the precise three-dimensional structure that these particles assume. The structure of the ribosome is in turn determined by a myriad of chemical interactions among its protein and RNA components. The main objective of this research is to gain a better understanding of such interactions by determining the molecular architecture of some of the protein-RNA complexes that underlie the three-dimensional structure of the ribosomal particle. The results will help to explain the manner in which the structure of the ribosome contributes to its function in protein synthesis.

9908399

Abstract

This project involves the upgrade of a 500 MHz NMR spectrometer to provide state-of-the-art capabilities in order to support the research of a group of users in the Department of Chemistry at University of Massachusetts, Amherst. The major research projects will be facilitated by the availability of the new instrumentation. This research will include determination of the structure of the peptide-binding domains of the molecular chaperones DnaK and BiP; determination of bound conformation of signal sequences upon interaction with their receptors; determination of the structure of an SRP-binding RNA fragment alone and in complex with a polypeptide; studies of the mechanism of folding of the predominantly beta-sheet protein, cellular retinoic acid biding protein; structural analysis of myoglobin mutants as models for guanylate cyclase; conformational studies of model helical peptides; determination of the active site geometry of metalloproteins; determination of the structure and dynamics of membrane proteins, including E. coli chemotactic receptors and colicin A; studies of domains of the E. Coli chemotactic receptor that are involved in adaptation; and determination of the structures of ribosomal proteins and the RNAs to which they bind. Additional projects will further utilize the remaining time on this instrument.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to obtain a detailed description of ligand-ribosome interactions during peptide bond formation, based on the hypothesis that the ribosome is a dynamic entity whose moving parts promote translation. Although recent high-resolution cryo-EM and crystallographic images provide new insights into the mechanism of translation, these 'snapshots' do not explain how the ribosome interacts with its numerous ligands or how structural changes in the ribosome facilitate this process. The specific objectives of this proposal are to define the structural and functional relationships between tRNA and the components of the peptidyl transferase center (PTC) and the exit site (E site) of the E. coil 50S ribosomal subunit, using chemical, biochemical and genetic approaches. (1) Putative differences in the structural organization of the PTC of archaeal and (eu)bacterial ribosomes, as well as possible changes in the position of tRNA relative to 23S rRNA during peptidyl transfer, will be examined by qualitative and quantitative analysis of the pattern of short-range, photochemically-induced crosslinks to the PTC from the 3' nucleotide of tRNA at the P and A sites and a transition-state analog that spans both. (2) Crystallography and tRNA crosslinking have revealed that there is a protein, L27, at or near the PTC of the (eu)bacterial 50S ribosomal subunit, a region that is otherwise composed entirely of RNA. Moreover, deletion of L27 leads to severe defects in translation. The functional role of L27 will be assessed by investigating the effects of specific truncations on cell growth and ribosome activity, and the site of tRNA crosslinking in L27 will be determined. (3) Protein L1 and its associated RNA comprise a flexible and semi-autonomous domain within the 50S subunit that appears to promote the release of deacyated tRNA from the ribosome following peptide bond formation. To test this theory, the ability of tRNA to interact with L1- RNA fragement complexes will be checked in vitro and the the effects of specific mutations in L1 and the 23S rRNA on tRNA release will be investigated in vivo. (4) As the main contribution of 23S rRNA to the catalysis of peptide bond formation may be to correctly position the peptidyl- and amnioacyl-tRNA substrates, mutations in the 23S rRNA at the PTC may disrupt this process. This hypothesis will be tested by evaluating the pattern of crosslinks between the 3' ends of P- and A-site tRNA and ribosomes containing mutant 23S rRNA. As many antibiotics target the ribosome, a detailed understanding of protein synthesis will contribute to the rational development of new antibiotics which are urgently needed in the face of widespread antibiotic resistance among pathogenic microorganisms.

A meeting devoted to recent research on ribosomes and the mechanism of protein synthesis, "Ribosomes: Form and Function," will be held at the Sea Crest Resort and Conference Center in North Falmouth, MA, from the evening of Sunday, June 3 through the evening of Thursday, June 7, 2007. It has been five years since the last meeting on ribosome structure and function, and the field has evolved significantly in the intervening years. Just a few years ago, none of the ongoing experiments were designed with the three-dimensional structure of the ribosome in mind, nor were data interpreted in detailed structural terms. Today, everything being done in the field refers to the particle's structure in one way or another. It is now time to review recent progress in the field and develop new strategies for the future.
The Organizers have established a Program Committee and, together, have selected 21 speakers prominent in the ribosome field who have all agreed to attend the meeting and make presentations. A further 20-25 speakers will be selected on the basis of abstracts submitted by registrants before the deadline of March 15, 2007. Sessions will be devoted to tRNA and Decoding, Translation Initiation, Peptide Bond Formation, Translocation, Termination and Recycling, Folding and Secretion of Nascent Polypeptides, Translational Control, and Biogenesis and Assembly of Ribosomes. Information on structure and function will be presented in all sessions, as will data from prokaryotic and eukaryotic systems.
The Organizers wish to enable as many young scientists as possible to participate in this meeting. Accordingly, funding is requested to support ten $500 travel awards for graduate students, postdoctoral scientists and junior faculty, with special consideration given to women and under-represented minorities. Specialized meetings of this sort are vital for the dissemination and exchange of current experimental results, new hypotheses and ideas for further growth. Although participants at all career stages benefit from such activities, they are particularly important for young scientists who will be able to meet, discuss their work, and establish collaborations with their peers as well as more senior scientists from the U.S. and abroad.