Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.
E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.
PBSX, a defective Bacillus subtilis prophage, maps to the metA-metC region of the chromosome. DNA (33 kilobases) from this region of the chromosome was cloned and analyzed by insertional mutagenesis with the integrating plasmid pWD3. This plasmid had a promoterless a-amylase gene (amyL) that provided information on the direction and level of transcription at the site of integration. Transcription under the control of the PBSX repressor proceeded in the direction metA to metC over a distance of at least 18 kilobases. Electrophoretic analysis of proteins produced by different integrant strains upon PBSX induction and by fragments subcloned in Escherichia coli allowed the identification of early and late regions of the prophage. A set of contiguous fragments directing mutagenic integration suggested that the minimum size of an operon that encodes phage structural proteins is 19 kilobases. The adaptation of PBSX transcriptional and replicational functions to a chromosomally based, thermoinducible expression system is discussed.
PBSX is a phage-like bacteriocin (phibacin) of Bacilus subtilis 168. Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles. The particles cannot propagate the PBSX genome. The particles produced by this suicidal response kill strains nonlysogenic for PBSX. A 5.2-kb region which controls the induction of PBSX has been sequenced. The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf. Pcf is similar to known sigma factors and acts at the late promoter P which has been located distal to pcf. The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases.The defective bacteriophage PBSX of Bacillus subtilis 168 is a phage-like bacteriocin, or phibacin, with curious biological properties. PBSX lysogens exposed to DNA-damaging agents produce PBSX phage-like particles which kill other nonlysogenic Bacillus strains (25) by binding to and disrupting the cell wall. Each PBSX particle has a small head and a relatively long tail. Phage heads appear to contain randomly selected 13-kb segments of host DNA, which are not injected into susceptible cells. PBSX is therefore not propagated by these particles (25,26). PBSX appears to be a particulate bacteriocin which has evolved from a bacteriophage.Induction of PBSX is controlled by the repressor gene xre (6,27,40,41). The amino acid sequence of Xre predicts that it is a helix-turn-helix (HLH) protein resembling other DNAbinding proteins such as lambda cI and Cro (14, 41). However, little is known about the mechanism of action of Xre, and apart from the suggestion that xre and a neighboring gene, now called open reading frame 10 (ORF10), are regulated by Xre itself, nothing is known about how the late genes are regulated (41).All known structural and lytic protein genes have been shown to be clustered within a large operon of at least 19 kb in length, which appears to be expressed from a single late promoter region called PL (Fig. la) (40). A number of mutations in regulatory genes, including xin (noninducible for PBSX) and xhi (heat inducible for PBSX), are located proximal to mutations affecting phage head and tail proteins such as xhd, xtl, and xki (6, 38). A regulatory mutation, xhi-1479, renders PBSX thermoinducible (6). A 1.2-kb EcoRI fragment from a PBSX wild-type strain was found to complement the xhi-1479 mutation, and sequence analysis identified the xre gene as the site of the mutation (41). xhi-1479 is thus analogous to the cIts857 mutation of bacteriophage lambda (36 Media. B. subtilis and Escherichia coli were grown in LuriaBertani (LB) broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) or LB agar. For selection, media contained chloramphenicol (at 3 pug/ml for low-copy-number vectors and 5 jig/ml for higher-copy-number vectors) and kanamycin (5 pug/ml) for B. subtilis, or chloramphenicol (15 jig/ml) and ampicillin (100 pug/ml) for E. coli as appropriate. a-Amylase activity was detected by adding starch (...
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