Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites.
Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one RNA subunit, serves as an adaptor between the cytoplasmic protein synthetic machinery and the translocation apparatus of the endoplasmic reticulum. To begin constructing a functional map of the 7SL RNA component of SRP, we extensively mutagenized the Schizosaccharomyces pombe SRP7 gene. Phenotypes are reported for fifty-two mutant alleles derived from random point mutagenesis, seven alleles created by site-directed mutagenesis to introduce restriction sites into the SRP7 gene, nine alleles designed to pinpoint conditional lesions, and three alleles with extra nucleotides inserted at position 84. Our data indicate that virtually all single nucleotide changes as well as many multiple substitutions in this highly structured RNA are phenotypically silent. Six lethal alleles and eleven which result in sensitivity to the combination of high temperature and elevated osmotic strength were identified. These mutations cluster in conserved regions which, in the mammalian RNA, are protected from nucleolytic agents by SRP proteins. The effects of mutations in the presumptive binding site for a fission yeast SRP 9/14 homolog indicate that both the identity of a conserved residue and the secondary structure within which it is embedded are functionally important. The phenotypes of mutations in Domain IV suggest particular residues as base-specific contacts for the fission yeast SRP54 protein. A single allele which confers temperature-sensitivity in the absence of osmotic perturbants was identified in this study; the growth properties of the mutant strain suggest that the encoded RNA is somewhat defective even at the permissive temperature, and is most likely unable to correctly assemble with SRP proteins at the nonpermissive temperature.
Liao
X
X
Department of Biochemistry, University of Illinois, Urbana-Champaign 61801.
Selinger
D
D
Althoff
S
S
Chiang
A
A
Hamilton
D
D
Ma
M
M
Wise
J A
JA
D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009690:Nucleic Acid Conformation; D009997:Osmotic Pressure; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D015138:RNA, Nuclear / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D018271:Signal Recognition Particle; D013696:Temperature
D012331:RNA, Fungal; D015138:RNA, Nuclear; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle