Identification of the RNA binding regions of SRP68/72 and SRP72 by systematic mutagenesis of human SRP RNA.
Within the large domain of the human signal recognition particle (SRP), 18 mutant SRP RNAs were constructed to disrupt Watson-Crick and G-U basepairs in helices 5, 6 and 8. Using a double-filter assay, the competitive binding of the mutant RNAs to purified human SRP68/72 or to a 7.4 kDa RNA-binding fragment of SRP72 (72frg) was measured. Binding of SRP68/72 was impaired by several mutations in the large domain with the most pronounced effects caused by changes in helix 5 (residues 222-231) and helix 8 (residues 176-191 and 202-214). Binding of the 72frg was diminished prominently by altering helix 5, in particular residues 120-128, and was unaffected by deleting helices 6 and 8. Deleting helix 8 diminished binding of SRP68/72 to a greater extent than deleting helix 6. The data suggest that nucleotide residues throughout most of the large SRP domain are directly and/or indirectly engaged in the binding of SRP68. In contrast, SRP72 binds only to a portion of the 5ef region.
Yin
Jiaming
J
Department of Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas, USA.
Iakhiaeva
Elena
E
Menichelli
Elena
E
Zwieb
Christian
C
D001665:Binding Sites / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000235:genetics / Q000378:metabolism*
D021122:Protein Subunits; C512269:SRP68 protein, human; C503636:SRP72 protein, human; D018271:Signal Recognition Particle; D012313:RNA