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Title:The Importance of RNA Sequence and Structure in the Assembly and Function of Signal Recognition Particle
Author(s):Selinger, David Alan
Doctoral Committee Chair(s):Wise, Jo Ann
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Molecular
Chemistry, Biochemistry
Abstract:Signal recognition particle (SRP), the ribonucleoprotein responsible for the recognition and targeting of nascent secretory proteins to the endoplasmic reticulum, is composed of 7SL RNA, along with six polypeptides. Earlier work in our lab identified a homolog of 7SL RNA in S. pombe, showed by gene disruption that it is essential for viability, and demonstrated by mutagenesis of the gene that a highly conserved structural element in the RNA, the domain IV tetranucleotide loop, is functionally important. By substituting UUCG for the wild type GAAA, I have demonstrated that the structure, not the sequence, of the tetraloop is critical. I used in vitro enzymatic probing to show that RNA molecules with lethal tetraloop mutations fail to effectively base pair the short helix adjacent to the tetraloop. Because evidence from other laboratories suggested that SRP54p might interact with domain IV of the RNA, I used these antibodies, directed against SRP54p, to investigate whether our domain IV mutants affect binding to this protein. Unlike the other five protein components of SRP, a binding site for SRP54p had not been mapped on the mammalian RNA by RNase protection experiments. SRP54p binding to mutant fission yeast RNAs was analyzed by native immunoprecipitation of the 7SL RNA with antibodies directed against SRP54p, followed by primer extension sequencing of the RNA. The results indicate that the structural and sequence specific elements whose importance I had previously demonstrated are required for SRP54p binding. Although the tetraloop is not directly involved in binding, the integrity of the adjacent short stem is critical, along with the three conserved residues proximal to the short stem that make up a sequence-specific recognition site for SRP54p. Native immunoprecipitation experiments on $\sp{35}$S-labeled cells demonstrate that the S. pombe particle, like canine SRP, consists of six proteins similar in size to their mammalian counterparts, complexed with the previously identified 7SL RNA. Mutations which are predicted to affect the binding of protein components other than SRP54p are suppressed by overexpression of this protein. The extent of phenotypic suppression correlates in an inverse fashion to the ability of these mutants to bind SRP54p under non-permissive conditions. (Abstract shortened by UMI.)
Issue Date:1993
Type:Text
Description:111 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
URI:http://hdl.handle.net/2142/72359
Other Identifier(s):(UMI)AAI9329158
Date Available in IDEALS:2014-12-17
Date Deposited:1993


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