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 targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.
The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle. To define the domain in Saccharomyces cerevisiae SRP
The signal recognition particle (SRP), a ubiquitous cytoplasmic ribonucleoprotein particle, plays an essential role in promoting co-translational translocation of proteins into the endoplasmic reticulum. Here, we summarise recent progress made in the understanding of two essential SRP functions: the signal recognition function, which ensures the specificity, and the elongation arrest function, which increases the efficiency of translocation. Our discussion is based on functional data as well as on atomic structure information, both of which also support the notion that SRP is a very ancient particle closely related to ribosomes. Based on the significant increase of knowledge that has been accumulating on the structure of elongation factors and on their interactions with the ribosome, we speculate about a possible mechanism of the elongation arrest function.
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