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.