In this award from the Chemistry of Life Processes in the Chemistry Division, Drs. Silvia Cavagnero and James Weisshaar, from the University of Wisconsin, Madison, will use single-molecule fluorescence microscopy to measure the time required for the biosynthesis of one single protein molecule by one ribosome. The timecourse of complex chemical and biophysical events in living cells, including gene expression, is poorly understood to date, largely due to the difficulties inherent in the interpretation of experiments performed in bulk solution. Towards the goal of the proposal, Drs. Cavagnero and Weisshaar will exploit unnatural amino acids bearing a fluorophore, and engineered to be incorporated very early in the sequence of specific genes of interest via single amber codons. This study will yield novel insights into the distribution of the translation rates of genes of interest within live bacterial cells. Upon incorporation into the nascent protein, the fluorophore-labeled unnatural amino acid will diffuse very slowly until release from the ribosome. After release of the fully synthesized protein carrying the fluorescent tag, diffusion of the fluorophore will be much more rapid. Quantitative analysis of individual camera frames will enable the determination of the residence time of the nascent protein on the ribosome with about one-second accuracy. The in vivo synthesis of the single-domain protein HmpH and the related three-domain protein Hmp will be studied first, to test and optimize the novel methodology. The translation rates of other multi-domain proteins may also be investigated. The ultimate goal is to develop a novel set of biological and spectroscopic tools to understand how pause sites affect the translation time of individual genes in live cells, and to learn how these pauses are distributed across different translating ribosomes. This novel approach is expected to open up opportunities for future studies of many other multi-step biochemical events in live cells, including folding, co-/post-translational modifications, and a wide variety of enzymatic reactions.
The time required for the stepwise buildup of proteins, i.e., the main components of living cells, is essential for the cell's well being and healthy function. If a protein is made too slowly it may never be able to contribute to cell's activity before the cell dies. Conversely, if a protein is made too fast it may never be able to achieve its correct three-dimensional shape because the fast synthesis does not allow enough time for proper three-dimensional folding. The protein may thus aggregate and irreversibly compromise the cell's function. In summary, it is clear that the timing of protein biosynthesis in the cell has to be just right, i.e., tightly regulated for correct cell function. The goal of this project is to measure the rate of protein synthesis in living bacterial cells, one molecule at a time. The single-molecule approach is the only way to measure synthesis times, given the complexity of the process in vivo. Each protein will be 'lit up' via labeling with a fluorescent tag directly in the living cell. This novel approach will open up unprecedented opportunities to explore the origin of cell's function and misfunction at high resolution. This study will thus enable Drs. Cavagnero and Weisshaar to interrogate the timecourse of one of the most important processes for life on earth, protein biosynthesis. This project will offer highly interdisciplinary opportunities to students and postdocs, who will synergistically integrate concepts from biology, fluorescence and microscopy, while carrying out this research. Drs. Cavagnero and Weisshaar will be very active in encouraging women, underrepresented minorities and undergraduate students to participate in this research and pursue careers in science. Together, they will also develop a new graduate recruitment event called CHOPS, standing for CHemistry OPportunitieS. A few college juniors and seniors interested in chemistry will come to visit the University of Wisconsin-Madison for a weekend in the fall. By providing hands-on single-molecule microscopy workshop for the visitors, the investigators will tie this research to the broader goal of enhancing underrepresented minority participation in science at the interface of chemistry and biology.
Proteins are the building blocks of cells and the engines of cellular growth and function. The central dogma of cell biology comprises the DNA code (specific for each protein type), the transcription of each code into mRNA messages by RNA polymerase, and the translation of the messages into specific proteins by the ribosomes. Much of our understanding of the details of these processes comes from careful biochemical studies in solution phase, in vitro. It is important to learn the degree to which inferences from solution phase studies apply to real, living cells. A revolution in fluorescence microscopy makes it increasingly feasible to monitor key transcription and translation steps in real time in live cells, at the level of single molecular events. Such studies open an entirely new window on the intimate details of biochemical processes in vivo. The goal of this research was to use fluorescence microscopy to directly measure the time scale of single translation events, i.e., the time required for biosynthesis of a one protein molecule by one ribosome in a live cell. We chose the bacterial system E. coli because it is easy manipulate the genetic content. We learned to use an electric current to place special fluorescent amino acid precursors into E. coli as well as a circle of DNA containing a special signal known as "amber codon" that codes for a fluorescent amino acid at the beginning of the sequence for a fluorescent protein product known as the green fluorescent protein, or GFP. Our goal was to use changes in diffusive behavior of single fluorescent labels inside living bacterial cells to monitor when the label becomes bound to a giant ribosome thereby moving slowly, marking the beginning of the translation event, and when the label becomes freely diffusing within the cellular space, after the protein has been fully synthesized and released from the ribosome, marking completion of the translation event. The experiments have not yet worked completely, but we made good progress that, we hope, will enable the Cavagnero group to carry the work forward in the future.
