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.