Genetic analysis of Schizosaccharomyces pombe 7SL RNA
Liao, Xiubei
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Permalink
https://hdl.handle.net/2142/19272
Description
Title
Genetic analysis of Schizosaccharomyces pombe 7SL RNA
Author(s)
Liao, Xiubei
Issue Date
1990
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, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one molecule of 7SL RNA, is required for translocation of secretory proteins into mammalian microsomal vesicles in vitro. In order to investigate the function of the RNA component of SRP, mutations were introduced into the S. pombe 7SL RNA gene by both site-directed and random mutagenesis.
The site-directed mutagenesis target was a 6-nucleotide segment within a 35-nucleotide structure which has been preserved in RNAs from many divergent organisms. This domain of 7SL RNAs interacts with two SRP proteins, the 19-kDa polypeptide and the 68/72-kDa heterodimer. The data demonstrated that, despite its conservation over vast evolutionary distances, many changes in this region can be tolerated under normal growth conditions. An exception is the lethality of several mutations at positions 159 and 160. Nucleotide 160 is the most highly conserved base in a single-stranded sequence which conforms to a consensus for the most common tetranucleotide loop in ribosomal RNAs. Mutations which are likely to affect the stability and/or conformation of the RNA give rise to a conditional phenotype: when osmolarity of the medium is raised, the RNAs become partially or completely defective in function at high temperature. The defects associated with mutations at position 159 can be partially rescued by mutating position 164 to restore base-pairing.
In order to obtain high efficiency random mutagenesis localized to the 7SL coding sequence, a new method for forced nucleotide misincorporation in vitro was developed. Greater than 50% mutants can be generated over a large (200 nucleotide) DNA segment. The results from random mutagenesis show that S. pombe 7SL RNA is generally insensitive to point mutations. Fifty seven mutant alleles, many with multiple substitutions, have been transformed into fission yeast. Only five of these, four of which are located in the 35-nucleotide conserved segment, generated lethal phenotypes. A mutant 7SL RNA carrying 6 point mutations shows a temperature sensitive-phenotype. Because of its slow response after shift to the nonpermisive temperature, the defect is likely to be in synthesis or assembly of SRP, rather than in its function.
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