Assembly of the human signal recognition particle (SRP): overlap of regions required for binding of protein SRP54 and assembly control.
Assembly of the human signal recognition particle (SRP) entails the incorporation of protein SRP54, mediated by a protein SRP1 9-induced conformational change in SRP RNA. To localize the region that controls this crucial step in the assembly of human SRP RNA, four chimeras, Ch-1 to Ch-4, composed of portions of human and Methanococcus jannashii SRP RNAs, were generated by PCR site-directed mutagenesis from a larger precursor. Protein-binding activities of the hybrid RNAs were determined using purified human SRP19 and a polypeptide (SRP54M) that corresponded to the methionine-rich domain of human SRP54. Mutant Ch-1 containing the large domain of M. jannashii SRP RNA, as well as mutant Ch-2 RNA in which helices 6 and 8 were replaced, bound SRP54M independently of SRP19. Mutant Ch-3 RNA, which contained M. jannashii helix 6, required SRP19 for binding of SRP54M, but mutant Ch-4 RNA, which possessed M. jannashii helix 8, bound SRP54M without SRP19. We concluded that the formation of a stable ternary complex did not rely on extensive conformational changes that might take place throughout the large domain of SRP, but was controlled by a smaller region encompassing certain RNA residues at positions 177 to 221. Five chimeric RNAs altered within helix 8 were used to investigate the potential role of a significant AA-to-U change and to determine the boundaries of the assembly control region. Reduced protein-binding activities of these chimeras demonstrated a considerable overlap of regions required for SRP54 binding and assembly control.
Yin
J
J
Department of Molecular Biology, The University of Texas Health Science Center at Tyler, 75708-3154, USA.
Yang
C H
CH
Zwieb
C
C
D001483:Base Sequence; D001665:Binding Sites; D002678:Chimera; D017931:DNA Primers; D006801:Humans; D017017:Methanococcus / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D012329:RNA, Bacterial; C069268:SRP54 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle