Analysis of essential structural and functional elements in the GTP-regulated signal recognition particle cycle
Althoff, Steven Martin
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Permalink
https://hdl.handle.net/2142/23012
Description
Title
Analysis of essential structural and functional elements in the GTP-regulated signal recognition particle cycle
Author(s)
Althoff, Steven Martin
Issue Date
1994
Doctoral Committee Chair(s)
Wise, Jo Ann
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Genetics
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
I have investigated signal recognition particle (SRP)-mediated protein targeting using a combination of genetic, biochemical, and molecular sequence analysis techniques. First, I consider the SRP cycle from the perspective of molecular evolution. This analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in the polypeptide subunits of the particle. The conservation of SRP cycle components, combined with biochemical data from the mammalian, bacterial and yeast systems, suggests that this pathway for protein export is ancient in evolutionary origin. Next, I have used a variety of genetic and biochemical techniques to define the role of the Srp54p GTPase in the SRP cycle. Repressing synthesis of the essential Srp54 protein produces a growth defect that correlates with an accumulation of secretory precursors. I have also analyzed the effects of 17 site-specific mutations in the G domain of Srp54p. Several mutant alleles confer lethal and conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of Srp54p. Enzymatic assays reveal that S. pombe Srp54p exhibits GTPase activity in vitro, while a mutant predicted to be catalytically defective has a reduced ability to hydrolyze GTP. Most importantly, the pattern of genetic dominance that these mutants display leads me to propose a model for the role of GTP hydrolysis by Srp54p during the SRP cycle, in which the SRP receptor $\alpha$ subunit (SR$\alpha$) serves as a GTPase activating protein (GAP) regulating signal sequence binding by the Srp54p subunit. Lastly, I have cloned and sequenced the S. pombe SR$\alpha$ gene. The gene encodes a 70 kDa protein that bears striking sequence similarity to the previously cloned mammalian and S. cerevisiae 70 kDa SR$\alpha$ proteins. The cloning of SR$\alpha$ opens the door to both exploring the biochemical effects of the SR$\alpha$ protein on the already existing Srp54p catalytically defective mutants and to genetically isolating the next downstream component in this complex GTPase cycle.
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