Determinants of a protein-induced RNA switch in the large domain of signal recognition particle identified by systematic-site directed mutagenesis.
Signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote the co-translational translocation of proteins across biological membranes. Human SRP RNA molecules exist in two distinct conformations, SR-A and SR-B, which may exchange during the assembly of the particle or could play a functional role in the SRP cycle. We have used systematic site-directed mutagenesis of the SRP RNA to determine the electrophoretic mobilities of altered RNA molecules, and we have identified the nucleotides that avert the formation of the two conformers. The conformer behavior of the various RNAs was addressed quantitatively by calculating a value zeta as an indicator of conformational variability. Single loose A-like forms were induced by changes in helix 5 [nucleotides (nt) at positions 111-128 or 222-231], helix 6 (nt at positions 141-150) and helix 7 (nt at position 169 and 170), whereas other mutations in helix 6 and helix 8 preserved the conformational variability of the mutant RNA molecules. The more compact B-like form was induced only when a small region (129-CAAUAU-134), located in the 5'-proximal portion of helix 6, was altered. Since this region is part of the binding site for SRP19, we suggest that protein SRP19 uses nucleotides at 129-134 to trigger the formation of the favored SR-B-form, and thus directs an early step in the hierarchical assembly of the large SRP domain.
Gowda
K
K
Department of Molecular Biology, The University of Texas Health Science Center at Tyler, Highway 271 N, PO Box 2003, Tyler, TX 75710, USA.
Zwieb
C
C
D001665:Binding Sites; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D018271:Signal Recognition Particle; D012313:RNA