Assembly of the human signal recognition particle (SRP): Overlap of regions required for binding of protein SRP54 and assembly control

Jiaming Yin, Ching Hui Yang, Christian Zwieb

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Assembly of the human signal recognition particle (SRP) entails the incorporation of protein SRP54, mediated by a protein SRP19-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.

Original languageEnglish (US)
Pages (from-to)1389-1396
Number of pages8
JournalRNA
Volume7
Issue number10
StatePublished - 2001
Externally publishedYes

Keywords

  • Assembly
  • RNA-protein interactions
  • RNP
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Molecular Biology

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