Protein-induced conformational changes of RNA during the assembly of human signal recognition particle.
The human signal recognition particle (SRP) is a large RNA-protein complex that targets secretory and membrane proteins to the endoplasmic reticulum membrane. The S domain of SRP is composed of roughly half of the 7SL RNA and four proteins (SRP19, SRP54, and the SRP68/72 heterodimer). In order to understand how the binding of proteins induces conformational changes of RNA and affects subsequent binding of other protein subunits, we have performed chemical and enzymatic probing of all S domain assembly intermediates. Ethylation interference experiments show that phosphate groups in helices 5, 6 and 7 that are essential for the binding of SRP68/72 are all on the same face of the RNA. Hydroxyl radical footprinting and dimethylsulphate (DMS) modifications show that SRP68/72 brings the lower part of helices 6 and 8 closer. SRP68/72 binding also protects the SRP54 binding site (helix 8 asymmetric loop) from chemical modification and RNase cleavage, whereas, in the presence of both SRP19 and SRP68/72, the long strand of helix 8 asymmetric loop becomes readily accessible to chemical and enzymatic probes. These results indicate that the RNA platform observed in the crystal structure of the SRP19-SRP54M-RNA complex already exists in the presence of SRP68/72 and SRP19. Therefore, SRP68/72, together with SRP19, rearranges the 7SL RNA in an SRP54 binding competent state.
Menichelli
Elena
E
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England.
Isel
Catherine
C
Oubridge
Chris
C
Nagai
Kiyoshi
K
D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D020733:RNA, Small Cytoplasmic / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry*; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000737:chemistry
C115990:7SL RNA; D020733:RNA, Small Cytoplasmic; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; D013463:Sulfuric Acid Esters; D017665:Hydroxyl Radical; C007482:dimethyl sulfate