Bacillus subtilis Ffh and scRNA are homologues of mammalian SRP54 and SRP RNA, respectively, which are components of the eukaryotic signal recognition particle (SRP). Ffh (446 amino acids) interacts with scRNA to form a stable complex in vivo. Here, we identified an RNA-binding domain of Ffh. The results obtained using a series of deletion mutants show that amino acid positions 364 to 432 in the C-terminal region of Ffh correlates with its ability to bind RNA. In mammals, the signal recognition particle (SRP) 1 plays an important role in targeting secretory proteins to the membrane of the endoplasmic reticulum. Mammalian SRP is a ribonucleoprotein particle composed of one RNA molecule (7 S RNA here referred to SRP RNA) and six polypeptides of 9, 14, 19, 54, 68, and 72 kDa (1, 2). SRP interacts with a signal sequence of a nascent polypeptide as it emerges from the ribosome, then the elongation of the nascent chain is inhibited (3, 4). The complex consisting of SRP, nascent polypeptide chain, and ribosome is targeted and binds to the heterodimeric SRP receptor in the endoplasmic reticulum membrane (3-5).The SRP54 subunit of SRP has a central function in recognizing and binding signal sequences (6 -8). This protein comprises the structurally distinct, N-terminal G-and C-terminal M-domains (9, 10). The G-domain contains three GTP-binding motifs, and it may play an essential role in mediating the interaction between SRP and the SRP receptor (11). The Mdomain is characterized by a high content of predominantly positively charged residues and an abundance of methionine residues. Moreover, there are three putative amphipathic helices in the M-domain (9). The M-domain is a binding site for the signal sequence and SRP RNA (12-15). The three-dimensional structure of SRP54 protein has been determined by scanning transmission electron microscopy (STEM) (16). The scanning transmission electron microscopy image consisting of one larger and one smaller domain (probably representing the Gand M-domain, respectively) joined by slender linker, was in agreement with genetic and biochemical data. A protein homologous to SRP54 has been identified in Eukaryotes and Eubacteria (3,(17)(18)(19)(20)(21)(22)(23)(24). The features of the two structural domains (G-and M-domains) of SRP54 are highly conserved in these proteins. Furthermore, these proteins form an SRP-like complex with RNA molecules that are structurally related to mammalian SRP RNA (25-28). The binding of Ffh to 4.5 S RNA, which are homologues of SRP54 and SRP RNA in Escherichia coli, respectively, is essential for the signal recognition function (29). Larsen and Zweib (30) proposed that SRP RNA consists of 8 helices (numbered 1 to 8). The nucleotide sequence in the region corresponding to the helix 8 is highly conserved among SRP RNA homologues, and this loop region mediates the binding to SRP54 and its homologues (17,31,32). The SRP RNA homologue of Bacillus subtilis scRNA (small cytoplasmic RNA) lacks helices 6 and 7, whereas those of E. coli 4.5 S RNA lack helices 1, 2, 3,...
A novel RNA species was isolated from Bacillus subtilis, and its sequence was determined and mapped to its genetic position. This RNA was termed BS190 RNA from the length of its mature form (190 nt), and the gene encoding it is located within the aspS-yrvM intergenic region of the B. subtilis genome. Northern blotting revealed that the novel RNA species is transcribed in vegetative cells as a larger precursor (BS201 RNA, 201 nt). After transcription, the 5' end of the precursor is processed to generate the mature form, BS190 RNA. A computer-aided prediction of the secondary structure of BS190 RNA showed that it can be folded into a single hairpin structure with some bulge structures. The authors found that the growth rate of a ∆BS190 mutant strain of B. subtilis was reduced when compared to the wild-type. A phylogenetic comparison of the sequence of the BS190 RNA gene with sequences from the databases suggests that RNA related to BS190 RNA appears to be encoded in the genomes of Bacillus halodurans and Listeria monocytogenes.
Escherichia coli cells contain abundant amounts of metabolically stable 4.5 S RNA. Consisting of 114 nucleotides, 4.5 S RNA is structurally homologous to mammalian 7 S RNA, and it plays an essential role in targeting proteins containing signal peptide to the secretory apparatus by forming an signal recognition-like particle with Ffh protein. It also binds independently to protein elongation factor G (EF-G) and functions in the translation process. This RNA contains a phylogenetically conserved RNA domain, the predicted secondary structure of which consists of a hairpin motif with two bulges. We examined the binding activity of mutants with systematic deletions to define the minimal functional interaction domain of 4.5 S RNA that interacts with EF-G. This domain consisted of 35-nucleotides extending from 36 to 70 nucleotides of mature 4.5 S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas those at A39, C40, C41, A42, G48, and G49 did not affect binding. These data suggested that the 10 nucleotides in 4.5 S RNA, which are conserved between 4.5 S RNA and 23 S rRNA, have a key role for EF-G binding. Based on the NMR-derived structure of mutant A47U, we further verified that substituting U at A47 causes striking structural changes and the loss of the symmetrical bulge. These results indicate the mechanism by which EF-G interacts with 4.5 S RNA and the importance of the bulge structure for EF-G binding.
Escherichia coli 4.5S RNA is a member of the signal recognition particle RNA family that binds to Ffh and EF-G proteins in vivo. To assess the binding affinity of E. coli 4.5S RNA, wild-type Ffh and a series of amino terminal truncated EF-G mutants with a histidine tag were over-expressed in Escherichia coli and purified. Among them, EF-G mutants with a deletion of all upstream sequences up to and including the second or the third GTP binding sequence element were expressed at high levels and bound with the same activity as wild-type EF-G. Nitrocellulose filter binding assays revealed that the binding affinity values (M 1a2 ) for Ffh and EF-G, defined as the concentration giving half-maximal binding, were 0.15 WM and 1.5 WM, respectively. Moreover, we also show that very little EF-G can form a stable complex with 4.5S RNA in vivo, whereas almost all Ffh binds to 4.5S RNA. ß
We isolated and characterized a novel small RNA from Bacillus subtilis. We termed this molecule BS203 RNA from the length of its mature form (203 nt) and located the corresponding gene at the yocI-yocJ intergenic region on the B. subtilis genome. Northern blotting revealed that it is transcribed in vegetative growing cells and that the amount of BS203 RNA decreased in the middle of the vegetative phase. A computer-aided prediction of the BS203 RNA secondary structure revealed three characteristic stem-loop structures. Despite active expression during the vegetative phase, growth of the knockout mutant was not affected by depletion of BS203 RNA. A phylogenetic comparison of the sequence of the BS203 RNA with other Bacillus species including B. cereus and B. halodurans C-125, or Clostridium perfringens suggests that the sequence is unique to Bacillus subtilis.
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