The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5′ domain. This creates the binding site for the Alu RNA 3′ domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3′ domain folding back by 180° onto the 5′ domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634–638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNAArg and C36 and G37 in tRNAAsp (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069–5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNAArg and tRNAAsp, the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899–4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different kcat and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.