GENETIC ANALYSIS TO THE SIGNAL RECOGNITION PARTICLE

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The signal recognition particle (SRP) is a ribonucleoprotein complex that is important for targeting exported proteins to the eukaryotic endoplasmic reticulum. Interestingly, homologues to components of the mammalian SRP and SRP receptor have recently been found throughout nature, including in Escherichia coli. E. coli homologues include products of the ffh, ffs, and ftsY genes. All of these genes are essential for E. coli viability, and biochemical evidence suggests that the products of these genes interact in vivo. Furthermore, some protein export defects are observed following depletion of the ffh and ftsY gene products. However, it is unclear how these proteins function in generalized protein export, nor is it known if they function in cellular processes apart from protein export. To determine the cellular function of the bacterial SRP the Ffh protein, a homologue to a 54 kD component of the eukaryotic SRP, will be studied by using a combination of genetic and biochemical approaches. E. coli strains have been constructed that permit depletion of Ffh from actively growing cells. These strains will be characterized by studying the effects of Ffh depletion on various cellular processes. Initially, experiments will he performed to determine if Ffh functions directly in bacterial protein export. This analysis will include the use of both in vivo and in vitro techniques to monitor efficiency of signal sequence processing and localization following Ffh depletion. It will also be determined if Ffh functions on the well characterized sec pathway of protein translocation. In addition, the possibility that Ffh is important for cellular processes other than protein export will be tested. Possible alternative activities for Ffh include roles in protein synthesis and protein folding. Conditional mutants of ffh will also be isolated. These mutants will, likewise, be characterized for defects in protein export, as well as in other cellular processes. The study of new ffh mutants will provide an independent way to determine the cellular function of its gene product. In addition, other classes of ffh mutants will also be sought that should provide new insights into the in vivo function of Ffh. The outcome of this research will be to understand how the SRP homologue Ffh functions in bacteria. Since Ffh is an essential protein, it is clear that it plays an important role in key cellular processes. By determining the in vivo role of this protein we will increase our understanding of fundamental cellular processes common to all life. If this homologue indeed functions in protein export then new insights will he gained into how proteins exit the cytoplasm. Furthermore, because of the highly conserved features of Ffh throughout nature, a better understanding of how the SRP functions in eukaryotes may also result. A potential application of this research may be to identify new targets for antimicrobial agents and to develop new technologies for more efficient export of medically important proteins.