Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components.We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.Signal recognition particle (SRP) l has been shown to couple the cytoplasmic protein synthesis machinery with the membrane-bound protein translocation machinery of the endoplasmic reticulum. The function of SRP is well established and can be readily assayed in vitro. Experiments performed in a wheat germ in vitro translation system that was supplemented with purified SRP and SRP-depleted microsomal membranes, and programmed with total pituitary RNA, coding primarily for the secretory protein preprolactin, and/or total reticulocyte RNA, coding primarily for the cytoplasmic protein globin, have led to the following model for SRP activity (called the "SRP cycle;" for review see reference 1). First, SRP binds with high affinity to ribosomes that are synthesizing secretory proteins (2) and arrests their synthesis at a specific point in the nascent chain (3), corresponding to that point where the signal sequence is fully exposed on the surface of the ribosome. Second, SRP interacts with its own Abbreviations used in this paper." DTT, dithiothreitol; 7MEG, 7-methylguanosine-5'-monophosphate; NaPi, sodium phosphate, pH 7.5; PMSF, phenylmethylsulfonylfluoride; SRP, signal recognition particle; TEA, triethanolamine/HOAc (pH 7.5, adjusted at room temperature). receptor in the endoplasmic reticulum, called SRP receptor (4, 5) or docking protein (6, 7). This interaction results in a release of the translation arrest (5, 6), and a weakening of the high affinity binding between SRP and the ribosome (8). The nascent chain then traverses the membrane co-translationally (9, 10); several models have been proposed for the mechanism of the passage (l 1-13), but the details remain unclear. After the formation of the functiona...

Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts. Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function. Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref. 6). 7SL RNA is composed of both unique and Alu-like sequences. SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively). The RNA moiety is required for SRP activity, as well as for structural integrity of the particle. To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences. We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein. In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis. Re-addition of the 9/14K protein did not restore the elongation arrest. We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

We have used the nuclease a-sarcin to map the binding sites of the 19-kDa and the 68/72-kDa proteins of signal recognition particle (SRP) on SRP RNA. We found that the regions of protection to nuclease afforded by the two proteins were distinct. p19 protected primarily the two tips in the RNA secondary structure. p68/72 protected a large region extending across the center of the particle and altered the nuclease pattern in the regions that p19 would bind, suggesting that these two proteins may be in dose proximity in the particle. The protection afforded by the two proteins in combination was equal to the sum of the individual protections. We have not observed cooperativity in the binding of these two proteins as assessed by the protection assay; nor do we have any evidence that the structure becomes more compact as it assembles. The map derived from this "footprint" analysis places the signal recognition domain (p54 bound to the RNA via the 19-kDa protein) and the elongation arrest domain (associated with the Alu end of the particle) on opposite ends of the particle. Thus, it is possible that SRP recognizes signals by the direct interaction of p54 with the signal sequence at the nascent chain exit site and simultaneously blocks elongation by the entrance of p9/14 into the aminocyl tRNA site 16 nm away.

We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP). In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S. pombe and 270 nucleotides in Y. lipolytica) present in postribosomal salt extracts of the cytoplasm. The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S. pombe and Y. lipolytica, respectively. Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase HI promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded. Both of the yeast RNAs bind under stringent conditions to canine SRP proteins. Most importantly, RNase protection of the S. pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements ofthe mammalian and yeast RNA. Taken together these data suggest strongly that we have identified yeast SRP homologues.Fractionation of in vitro systems that mimic the translocation of nascent secretory proteins across the membrane of the mammalian endoplasmic reticulum (ER) has led to the identification ofa soluble ribonucleoprotein (RNP), the signal recognition particle (SRP), which is essential for targeting of nascent secretory proteins to the ER membrane. SRP is thought to function as an adapter between the cytoplasmic translation and the ER membrane-bound protein translocation machineries (1).SRP is a biochemically well-defined entity. It contains one RNA of 300 nucleotides (nt) (7SL RNA, here referred to as SRP RNA) and 6 polypeptides (2, 3). The canine SRP proteins can be separated from the RNA in native form and rebound to the RNA to reconstitute active SRP (4, 5). Studies on perturbed SRPs reconstituted either with modified SRP proteins or with only a subset of the proteins allowed us to dissect the functional contributions of individual SRP proteins (5,6). Heterologous reconstitutions of the canine proteins with Xenopus or Drosophila SRP RNAs also yield active particles in the in vitro assay, thus demonstrating the evolutionary conservation of SRP RNA (4). Phylogenetic analysis of the sequences from the human, amphibian, and insect RNAs has defined the secondary structure (7,8). The binding sites of two of the proteins (p19, a monomeric protein, and p68/72, a heterodimer) have been mapped (9) onto this structure.As a first step toward elucidating SRP function in vivo in genetically tractable organisms we have identified homologues of SRP in the yeasts Schizosaccharomyces pombe and Yarrowia lipolytica. Our strategy involved fractionation of yeast extracts to enrich for RNPs of similar b...

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