Cooperative assembly of signal recognition particle RNA with protein SRP19.
Signal recognition particle (SRP) is a ribonucleoprotein complex involved in the targeting of secretory proteins to the lipid bilayer of the endoplasmic reticulum. SRP contains the protein SRP19, which is an important structural and functional component, believed to promote the assembly of the particle. We have purified the human SRP19 protein to homogeneity from recombinant bacteria which overexpress the polypeptide, and have studied details of the binding to SRP RNA via gel mobility shift and RNase sensitivity assays. SRP19 interacts with two SRP RNA conformers with different affinities such that the more compact RNA species is bound more avidly. Furthermore, binding was found to be highly cooperative. Binding constants and Hill coefficients were determined for several mutant SRP RNAs in which individual RNA helices were deleted. These results confirmed that both SRP RNA helices 6 and 8 are important for SRP19 binding. Enzymatic RNA structure probing of a 150-nucleotide mutant SRP RNA fragment and of the corresponding RNA-SRP19 complex showed that cooperativity may be due to protein-induced conformational changes in the large domain of the SRP RNA. Finally, SRP19 bound specifically not only to SRP RNA but also to the A-form of Escherichia coli 5S ribosomal RNA, thereby indicating structural similarities between these two RNA molecules.
Walker
K P
KP
Department of Molecular Biology, University of Texas Health Science Center at Tyler 75710, USA.
Black
S D
SD
Zwieb
C
C
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D018271:Signal Recognition Particle; D012313:RNA