This project focuses on one of the most fundamental of cellular processes: protein synthesis by the ribosome, a universally conserved ribonucleoprotein enzyme. The ultimate goal in the Gonzalez laboratory is to characterize the conformational dynamics of the ribosome and other biomolecules involved in protein synthesis and utilize these data to elucidate the detailed molecular mechanism of protein synthesis. Novel fluorescence spectroscopic approaches are used to study the ribosomal molecular machine at the single-molecule level, focusing on conformationally dynamic regions of the biomolecule whose functional importance remains largely mysterious. Using single-molecule fluorescence, the mechanics of conformational changes within the ribosome are identified, quantified and coupled to biochemical steps in the overall process of protein synthesis. Achieving this level of resolution, however, requires merging the areas of molecular biology, biochemistry, biophysics, optics, microscopy, surface chemistry, microfluidics, and statistical data analysis. The laboratory is therefore highly interdisciplinary and is actively engaged in complementary technological developments, such as the application of microfluidic design principles to the sample cells used for single-molecule fluorescence measurements and the development of mathematically robust statistical models for data analysis that will expand the capabilities of single-molecule fluorescence spectroscopies. Beyond exclusive impact in the area of protein synthesis, the general principles developed by this research are directly applicable to the study of the numerous complex biochemical reactions that are of critical functional importance to the cell. The interdisciplinary nature of the research in the Gonzalez laboratory provides a unique opportunity to integrate students of varied scientific backgrounds and stimulate cross-discipline training. In order to achieve similar educational impact in the broader departmental and university communities at Columbia University, Prof. Gonzalez is integrating nucleic acid and single-molecule biophysics into the curriculum, participating in the design of a shared molecular biology facility, and organizing a discussion forum for users of this facility. Prof. Gonzalez has also put in place a long-term undergraduate/high-school student research program composed of projects to be exclusively executed by undergraduate and high-school student researchers. This educational and practical experience in all aspects of an interdisciplinary research project will prepare students for future work in contemporary scientific laboratory settings. This project is cofunded by the Divisions of Molecular and Cellular Biology and Chemistry.
The ribosome is the molecular machine that is responsible for making all of the proteins that living cells require to function. The main goal of this project was to develop and apply new approaches that enable the mechanical process through which ribosomes make proteins to be directly observed and quantified. By combining genetic, biochemical, and chemical approaches with state-of-the-art fluorescence microscopy, we have directly monitored individual ribosomes as they undergo the mechanical process of making a protein. Based on a number of previously published studies, we expected that some of the most important steps necessary to make a protein would require parts of the ribosome to undergo very large movements. The approaches that we developed and applied in this study allowed us to directly observe these movements for the first time. Surprisingly, we discovered early on in this project that some of the most important movements that ribosomes must undergo as they make a protein are driven by random thermal motions. Expanding from this fundamental discovery, we then sought to determine how these random thermal motions of the ribosome could be "organized" in such a way so as to allow the ribosome to perform the complex mechanical task of making a protein. By pursuing this line of inquiry, we learned that the structure of the ribosome itself has evolved to limit, preferentially bias, and coordinate these random thermal motions. In addition, we learned that the structures of the transfer RNA substrates that the ribosome uses to make proteins as well as the interactions that the transfer RNAs make with the ribosome help to further guide the random thermal motions of the ribosome. Most importantly, however, we learned that protein cofactors that are essential for enabling ribosomes to make proteins exert their power by robustly influencing and regulating the random thermal motions of the ribosome. As a result of these studies, we have a much deeper and more detailed understanding of the mechanical processes through which ribosomes make proteins. This understanding provides a framework for investigating how healthy cells regulate the process through which ribosomes make proteins; how cancerous, virally infected, or otherwise unhealthy cells lose the ability to regulate the process through which ribosomes make proteins; how antibiotic drugs that specifically target bacterial ribosomes block the ability of bacterial ribosomes to make proteins; and how new drugs might be developed that allow researchers and doctors to control the ability of ribosomes to make proteins in order to treat serious human diseases.
