IDENTIFICATION OF A MINIMAL ALU RNA FOLDING DOMAIN THAT SPECIFICALLY BINDS SRP9 14/

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRP Phi 14-9. Recombinant SRP Phi 14-9 i s a fusion protein containing on a single polypeptide chain the sequen ces of both the SRP14 and SRP9 proteins that are part of the Alu domai n of the signal recognition particle (SRP). SRP Phi 14-9 has been show n to bind to the 7SL RNA of SRP and it confers elongation arrest activ ity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3 ' ends were produced in vitro using ribozyme technology and tested for specific SRP Phi 14-9 binding in a quantitative equilibrium competiti on assay. This enabled identification of an Alu RNA of 86 nt (SA86) th at competes efficiently with 7SL RNA for SRP Phi 14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two st em-loops that are connected by a highly conserved bulge and, in additi on, a part of the central adaptor stem that contains the sequence at t he very 3' end of 7SL RNA. Circularly permuted variants of SA86 compet ed only if the 5' and 3' ends were joined with an extended linker of f our nucleotides. SASS can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for spec ific recognition by SRP Phi 14-9. These results suggest that Alu RNA i dentity is determined by a characteristic tertiary structure, which mi ght consist of two flexibly linked domains.