EM OF POLYSOME FORMATION DURING MEMBRANE PROTEIN SYNTHESIS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a cell-free protein synthesis system which produces high yields of membrane proteins in E.coli inner membrane vesicles. These membrane proteins are inserted into the vesicles co-translationally through docking of the ribosomes to translocon channels embedded in the membranes. We would like to verify that, analogous to soluble protein synthesis, multiple membrane protein-producing ribosomes can translate from a single mRNA and that these ribosomes will each be docked to individual translocons. Ultimately, we would like to use cryoEM to image these polysomes docked to our vesicle membranes. The mechanisms involved in membrane protein synthesis are currently an active area of research. Though polysome formation has been hypothesized for membrane proteins, to the best of our knowledge they have not been imaged directly. Our cell-free synthesis system provides many advantages previously unavailable for polysome studies. First, the reaction occurs in an open environment allowing direct access to the polysome structures without harsh cell lysis procedures that might damage fragile polysomes. Second, the system produces only the single desired target protein, so results are not obscured by polysomes synthesizing unrelated soluble proteins. Thus, we believe cell-free technology provides a more optimal platform for imaging these polysomes.