Role of SRP19 in assembly of the Archaeoglobus fulgidus signal recognition particle.
Previous studies have shown that SRP19 promotes association of the highly conserved signal peptide-binding protein, SRP54, with the signal recognition particle (SRP) RNA in both archaeal and eukaryotic model systems. In vitro characterization of this process is now reported using recombinantly expressed components of SRP from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidis. A combination of native gel mobility shift, filter binding, and Ni-NTA agarose bead binding assays were used to determine the binding constants for binary and ternary complexes of SRP proteins and SRP RNA. Archaeal SRP54, unlike eukaryotic homologues, has significant intrinsic affinity for 7S RNA (K(D) approximately 15 nM), making it possible to directly compare particles formed in the presence and absence of SRP19 and thereby assess the precise role of SRP19 in the assembly process. Chemical modification studies using hydroxyl radicals and DEPC identify nonoverlapping primary binding sites for SRP19 and SRP54 corresponding to the tips of helix 6 and helix 8 (SRP19) and the distal loop and asymmetric bulge of helix 8 (SRP54). SRP19 additionally induces conformational changes concentrated in the proximal asymmetric bulge of helix 8. Selected nucleotides in this bulge become modified as a result of SRP19 binding but are subsequently protected from modification by formation of the complete complex with SRP54. Together these results suggest a model for assembly in which bridging the ends of helix 6 and helix 8 by SRP19 induces a long-range structural change to present the proximal bulge in a conformation compatible with high-affinity SRP54 binding.
Diener
J L
JL
Department of Biology and Center for the Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, California 95064, USA.
Wilson
C
C
D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D020733:RNA, Small Cytoplasmic / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism
C115990:7SL RNA; D019843:Archaeal Proteins; D012329:RNA, Bacterial; D020733:RNA, Small Cytoplasmic; D016601:RNA-Binding Proteins; C117074:SRP19 protein, human; C069268:SRP54 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; D004047:Diethyl Pyrocarbonate