No abstract

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

The signal recognition particle (SRP) plays a central role in directing the export of nascent proteins from the cytoplasm of mammalian cells. An SRP-dependent translocation machinery in bacteria has not been demonstrated in previous genetic and biochemical studies. Sequence comparisons, however, have identified (i) a gene in Escherichia coli (ffh) whose product is homologous to the 54-kilodalton subunit (SRP54) of SRP, and (ii) an RNA encoded by the ffs gene (4.5S RNA) that shares a conserved domain with the 7SL RNA of SRP. An antiserum to Ffh precipitated 4.5S RNA from E. coli extracts, implying that the two molecules reside in a complex. The 4.5S RNA can also bind to SRP54 and can replace 7SL RNA in an enzymatic assay. The product of a dominant mutation in the ffs gene (4.5S RNAdl1) is also coprecipitated by the antiserum to Ffh protein and is lethal when expressed from an inducible promoter. After induction of 4.5S RNAdl1, the earliest observed phenotype was a permanent induction of the heat shock response, suggesting that there was an accumulation of aberrant proteins in the cytoplasm. Late after induction, translocation of beta-lactamase was impaired; this may be an indirect effect of heat shock, however, because translocation of ribose binding protein or of the porin, OmpA, was unaffected. An unusual separation of the inner and outer membranes, suggestive of a defect in cell envelope, was also observed. Protein synthesis did not cease until very late, an indication that 4.5S RNA probably does not have a direct role in this process.

The Alu domain of the mammalian signal recognition particle (SRP) comprises the heterodimer of proteins SRP9 and SRP14 bound to the 5' and 3' terminal sequences of SRP RNA. It retards the ribosomal elongation of signal-peptide-containing proteins before their engagement with the translocation machinery in the endoplasmic reticulum. Here we report two crystal structures of the heterodimer SRP9/14 bound either to the 5' domain or to a construct containing both 5' and 3' domains. We present a model of the complete Alu domain that is consistent with extensive biochemical data. SRP9/14 binds strongly to the conserved core of the 5' domain, which forms a U-turn connecting two helical stacks. Reversible docking of the more weakly bound 3' domain might be functionally important in the mechanism of translational regulation. The Alu domain structure is probably conserved in other cytoplasmic ribonucleoprotein particles and retroposition intermediates containing SRP9/14-bound RNAs transcribed from Alu repeats or related elements in genomic DNA.

Alu elements are the most abundant repetitive elements in the human genome; they emerged 65 million years ago from a 5′ to 3′ fusion of the 7SL RNA gene and amplified throughout the human genome by retrotransposition to reach the present number of more than one million copies. Over the last years, several lines of evidence demonstrated that these elements modulate gene expression at the post-transcriptional level in at least three independent manners. They have been shown to be involved in alternative splicing, RNA editing and translation regulation. These findings highlight how the genome adapted to these repetitive elements by assigning them important functions in regulation of gene expression. Alu elements should therefore be considered as a large reservoir of potential regulatory functions that have been actively participating in primate evolution.