Analysis of essential structural and functional elements in the GTP-regulated signal recognition particle cycle

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 α subunit (SRα) serves as a GTPase activating protein (GAP) regulating signal sequence binding by the Srp54p subunit. Lastly, I have cloned and sequenced the S. pombe SRα gene. The gene encodes a 70 kDa protein that bears striking sequence similarity to the previously cloned mammalian and S. cerevisiae 70 kDa SRα proteins. The cloning of SRα opens the door to both exploring the biochemical effects of the SRα protein on the already existing Srp54p catalytically defective mutants and to genetically isolating the next downstream component in this complex GTPase cycle.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Althoff, Steven Martin

Contributors dc:contributor

• Wise, Jo Ann

• Biology, Genetics
• Biology, Microbiology
• Chemistry, Biochemistry

Statement dc:rights

• Copyright 1994 Althoff, Steven Martin

Language dc:language

eng

Identifier

AAI9512284
(UMI)AAI9512284

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/23012