The Mechanism by which Ribosomes Form Proteins

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Hardesty 9315034 Folding of nascent proteins into a three-dimensional conformation as they are synthesized linearly on ribosomes will be studied. The role of the ribosome and the chaperones in the folding process will be evaluated. Three enzymes (bacterial dihydrofolate reductase, chloramphenicol acetyl transferase, and mammalian rhodanese) will be synthesized by coupled transcription/translation with ribosomes, tRNA and enzymes derived from Escherichia coli in a cell-free system. Folding of the nascent peptides as they are elongated by the sequential, C-terminal addition of amino acids to peptidyl-tRNA on ribosomes will be monitored by florescence techniques. Acquisition of enzymatic activity will be used as a measure of correct folding into the native conformation. For studies involving florescence, a fluorophore such as coumarin will be incorporated at a specific site in the nascent peptide from the corresponding amino acyl-tRNA derivative, such as N(coumarin)-Mer- tRNA for incorporation of coumarin at the N-terminus of the nascent protein. Special emphasis will be given to testing the hypothesis, and to a determination of what part of this folding, if any, takes place within a tunnel or cavity in the large ribosomal subunit. The role of chaperones in folding of the three enzymes also will be studied. %%% A detailed understanding of the molecular forces and processes that are involved in folding of a linear peptide into the specific, three dimensional conformations of a protein in its native state is one of the most fundamental and challenging problems currently under intensive investigation in molecular biology. This process is crucial for the formation of active, soluble enzymes rather than aggregation of newly formed proteins into insoluble, intractable mass of denatured protein as occurs with egg white when it is cooked. An understanding of the process is critical for the rational design and synthesis of new enzymes. Understanding of the folding process would be a large step toward developing the theory and procedure that will make it possible to produce new protein catalysis for research, medicine and industry, many of which may not occur in living cells. Largely because of the inherent problems and technical difficulties, most studies have focused on refolding of proteins from their denatured state. Our approach is fundamentally different. Proteins are synthesized linearly on ribosomes by the sequential addition of amino acids from their N- terminal to C-terminal ends. We use florescence techniques to monitor folding of nascent proteins as they are formed. Techniques have been developed by which we covalently attached a fluorophore to a specific amino acid as aminoacyl-tRNA then incorporated the derivatized amino acid at a unique point in a nascent protein as it is formed on a ribosome. Fluorescence from the fluorophore is used to monitor synthesis and folding of the nascent protein. The role of the ribosome itself and "chaperone" proteins in the folding process will be studied. ***