| Popis: |
We introduce Protein Synthesis Monitoring (PSM) – a technique to monitor protein synthesis in live cells. In PSM, we transfect cells with tRNA labeled as FRET donors and acceptors. A FRET signal is generated only when a donor- and an acceptor-labeled tRNA come in close contact (< 7nM), as they do on the ribosome during elongation. The intensity of the FRET signal correlates with the number of ribosomes engaged in protein synthesis, providing a real-time, live-cell assay for measuring rates of protein synthesis. PSM can be used to monitor the rate of either total protein synthesis (overall PSM), using bulk tRNAs, or of the synthesis of a specific protein (specific PSM), using specific pairs of tRNA to mark the protein of interest. PSM has sub-micron spatial and sub-second temporal resolutions. Cells continue to live and grow normally, and the synthesized proteins are unchanged since the labeling is on the tRNA itself and not on the amino acid. We have demonstrated specific PSM for monitoring synthesis of a viral protein during viral infection using Isoleucine tRNA, and for monitoring synthesis of collagen during fibrosis in mouse fibroblasts using tRNA-Gly and tRNA-Pro, as collagen is distinguished by many repeats of Gly-Pro dipeptides. We will discuss these results as well as additional applications of PSM in basic research, drug discovery, cell sorting, neurobiology, cancer, biomanufacturing, viral infections, and various protein-synthesis specific diseases. |
