title: Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein.
abstract: Drosophila P-element somatic inhibitor protein (PSI) regulates splicing of the P-element transposase pre-mRNA by binding a pseudo-splice site upstream of the authentic splice site using four tandem KH-type RNA binding motifs. While the binding domains and specificity of PSI have been established, little is known about the contributions of each PSI KH domain to overall protein stability and RNA binding affinity. Using a construct containing only the RNA binding domain of PSI (PSI-KH03), we introduced a physiologically relevant point mutation into each KH domain of PSI individually and measured stability and RNA binding affinity of the resulting mutant proteins. Although secondary structure, as measured by circular dichroism spectroscopy, is only subtly changed for each mutant protein relative to wild type, RNA binding affinity is reduced in each case. Mutations in the second or third KH domains of the protein are significantly more deleterious to substrate recognition than mutation of the outer (first and fourth) domains. These results show that despite the ability of a single KH domain to bind RNA in some systems, PSI requires multiple tandem KH domains for specific and high-affinity recognition of substrate RNA.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Nikolas H
authors.initials: NH
authors.lastname: Chmiel
authors.forename: Donald C
authors.initials: DC
authors.lastname: Rio
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D051860:Fragile X Messenger Ribonucleoprotein 1; D009687:Nuclear Proteins; D012313:RNA; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C467328:FMR1 protein, Drosophila; C092281:PSI protein, Drosophila