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Research Paper
Identification of the RNA Binding Regions of SRP68/72 and SRP72 by Systematic Mutagenesis of Human SRP RNA
Jiaming Yin, Elena Iakhiaeva, Elena Menichelli and Christian Zwieb
volume 4 | issue 3
july-decemberSubscribe to this journal for $59/year
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.
Authors
Jiaming Yin
Department of Molecular Biology; University of Texas Health Science Center at Tyler; Tyler, Texas USA
Elena Iakhiaeva
Department of Molecular Biology; University of Texas Health Science Center at Tyler; Tyler, Texas USA
Elena Menichelli
MRC Laboratory of Molecular Biology; Cambridge, England
Christian Zwieb
Department of Molecular Biology; University of Texas Health Science Center at Tyler; Tyler, Texas USA