A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle

Abstract

The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum. During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency. Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP. We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA. However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity. RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities. SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest. This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function. Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA. Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function.

The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure

“…In mammalian SRP, the proteins SRP14 and SRP9 bind specifically and exclusively as a heterodimeric complex to the Alu portion of 7SL RNA (Strub & Walter, 1990)+ In S. cerevisiae, a homolog of the mammalian SRP14 protein, Srp14p, has been identified, whereas a homolog of the SRP9 protein has not (Brown et al+, 1994) suggesting that Srp14p alone, possibly as a homodimer, may bind scR1 RNA+ This hypothesis was further strengthened by the finding that the mammalian proteins SRP9 and SRP14 were, despite their primary sequence divergence, structurally homologous (Birse et al+, 1997) suggesting that the heterodimer may have evolved from a homodimeric protein+ To test whether Srp14p can bind S. cerevisiae SRP RNA, scR1 RNA, we synthesized the protein and the RNA in vitro+ Both genes, SRP14 and SCR1, were am-plified from the yeast genome and inserted into plasmids to allow their transcription by SP6 and T7 RNA polymerase, respectively (see Materials and Methods)+ The synthetic transcripts comprising the SRP14 coding region were used to program wheat germ extract for the synthesis of [ 35 S]-labeled Srp14p+ The translation reaction was then incubated with in vitro-synthesized biotinylated scR1 RNA and the RNA-bound protein separated from free protein with immobilized streptavidin+ The bound protein was displayed by SDS-PAGE and visualized by autoradiography+ Reproducibly, ;40% of [ 35 S]-labeled Srp14p was bound to scR1 RNA, whereas ,5% of the protein was bound to a control RNA (Fig+ 1)+ The control RNA (cRNA) represents a portion of the antisense strand of the murine SRP14 mRNA and was previously used as a negative control in RNA-binding assays with the mammalian SRP9/14 heterodimer (Bovia et al+, 1994)+ The observed binding efficiency of Srp14p is practically identical to that found for SRP9/14 binding to Alu RNA (Bui et al+, 1997)+ Furthermore, increasing the scR1 RNA concentration by 10-fold and varying the salt concentrations between 150 mM and 350 mM did not change the binding efficiency, indicating that the binding conditions were optimal (results not shown)+ In addition, we tested an RNA that was derived from an scR1 RNA gene lacking the most 59 adenosine residue (results not shown)+ It bound Srp14p with the same efficiency as scR1 RNA, demonstrating that the adenosine is dispensable for protein binding+…”

“…The mammalian SRP has been used as a model for the extensive characterization of SRP functions+ It comprises six proteins and one RNA molecule (SRP or 7SL RNA)+ The SRP 54 protein specifically recognizes signal sequences and together with stem VIII of 7SL RNA is required for the targeting of the nascent chainribosome complex to the ER membrane (for review, see Lütcke, 1995)+ SRP54 and stem VIII of 7SL RNA represent the evolutionarily most highly conserved SRP structures and have been recognized in many organisms of all three kingdoms (for references, see Samuelsson & Zwieb, 1999)+ The 59 and 39 sequences of 7SL RNA that are homologous to the Alu family of repetitive sequences as well as two proteins, SRP9 and SRP14, constitute the Alu domain of mammalian SRP+ The Alu domain mediates a transient arrest in the elongation of nascent chains that increases the efficiency of translocation in vitro (Siegel & Walter, 1985;Thomas et al+, 1997; for a review, see Bui & Strub, 1999)+ SRP9 and SRP14 are structural homologs and members of the family of small a/b RNA-binding proteins (Birse et al+, 1997)+ Binding of SR9/14 to the Alu portion of 7SL RNA appears to induce conformational changes in the protein as well as in the RNA moieties (Janiak et al+, 1992;Bui et al+, 1997;Weichenrieder et al+, 1997)+ Such adaptive changes have been suggested to play a crucial role in the direct interaction between the Alu domain and the ribosome that affects elongation arrest (Thomas et al+, 1997)+…”

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

“…The potential flexibility of the Alu domain might be relevant for the elongation arrest function of SRP+ It would allow SRP to exist in clearly different states during its functional cycle and it would pose the crucial C-terminal tail of SRP14 (Thomas et al+, 1997;Mason et al+, 2000) into alternating structural environments+ In the context of the Alu retroposition intermediates repeated in tandem, the flexibility between the Alu RNP 59 and 39 domain combined with the high local particle concentration due to the covalent linkage could favor the formation of a compact particle that resembles the SA88 RNP dimer+ Such a particle could be of functional importance at some stage in the retroposition cycle+…”

Signal Recognition Particle Alu Domain Occupies a Defined Site at the Ribosomal Subunit Interface upon Signal Sequence Recognition

“…The components in SRP that mediate these additional contacts remain to be determined. The C-terminal region of SRP14, which is essential for elongation arrest activity (12), may contribute to mediating these contacts.…”

“…SRP9/14 and the 5′ and 3′ ends of SRP RNA form the Alu domain of SRP, which mediates elongation arrest activity. Elongation arrest is detected in vitro as a complete arrest or a transient delay in the elongation of the nascent chain, and its absence decreases the translocation efficiency (10)(11)(12). In vivo, it is required for the tight accommodation of the translation and the translocation processes (13).…”