Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S. cerevisiae is essential for growth

We have isolated and sequenced genes from Saccharomyces cerevisiae (SRP54SC) and Schizosaccharomyces pombe (SRP54sp) encoding proteins homologous to both the 54-kD protein subunit (SRP54mam) of the mammalian signal recognition particle (SRP) and the product of a gene of unknown function in Escherichia coli, ffh (Romisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Nature (Lond.). 340:478-482; Bernstein H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, P. Walter. 1989. Nature (Lond.). 340:482-486). To accomplish this we took advantage of short stretches of conserved sequence between ffh and SRP54mam and used the polymerase chain reaction (PCR) to amplify fragments of the homologous yeast genes. The DNA sequences predict proteins for SRP54sc and SRP54sp that are 47% and 52% identical to SRP54mam, respectively. Like SRP54mam and ffh, both predicted yeast proteins contain a GTP binding consensus sequence in their NH2-terminal half (G-domain), and methionine-rich sequences in their COOH-terminal half (M-domain). In contrast to SRP54mam and ffh the yeast proteins contain additional Met-rich sequences inserted at the COOH-terminal portion of the M-domain. SRP54sp contains a 480- nucleotide intron located 78 nucleotides from the 5' end of the open reading frame. Although the function of the yeast homologues is unknown, gene disruption experiments in S. cerevisiae show that the gene is essential for growth. The identification of SRP54sc and SRP54sp provides the first evidence for SRP related proteins in yeast.     

Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY

A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis SecY homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.     

Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis secY. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).     

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli.

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.     

Cloning and sequence analysis of hydrogenase regulatory genes (hydHG) from Salmonella typhimurium.

The nucleotide sequence of the hydHG operon, comprised of chromosomal genes that regulate labile hydrogenase activity in Salmonella typhimurium, was compared with the reported hydHG sequence of Escherichia coli. Nucleotide sequence analysis of a 4.8 kb EcoRI fragment of Salmonella chromosomal DNA revealed that one of the open reading frames (ORF) encoded a protein of 441 amino acid residues. This large ORF was identified on a 2.7 kb Eco RI/HindIII fragment and coded for the complete hydG gene. The carboxy-terminus (626 bp) of the hydH gene also was present immediately upstream of hydG. Expression of the Salmonella hydG gene in a T7 promoter/polymerase system revealed the presence of a unique 45 kDa protein band. The incomplete hydH gene was not expressed. It is proposed that the labile hydrogenase activity in S. typhimurium may be regulated by the multiple component system.     

Protein SRP54 of human signal recognition particle: cloning,expression, and comparative analysis of functional sites

Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes. An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA. To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells. Recombinant SRP54 was purified from both sources. The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin. Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction. A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54. Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.     

TLR序列在SRP54蛋白与SRPRNA和信号肽结合中的作用

SRP54蛋白是信号识别颗粒(signal recognition particle)的一个关键组分.对人SRP54蛋白328～330位的TLR3个氨基酸进行人工诱变,在大肠杆菌BL21(DE3)pLysS中表达了A3突变体,并对A3突变体进行纯化和Superdex75凝胶过滤分析.观察到A3突变体丧失了与SRPRNA结合的能力,其自身也不能形成二聚体.结果证明,TLR这3个氨基酸残基与二聚体结构的形成有关,TLR是SRP54蛋白结合SRPRNA和新生蛋白质信号肽所必需的关键性氨基酸序列.     

Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139

The cellulase gene from the alkalophilic Bacillus sp. strain 1139 was cloned in Escherichia coli using pBR322. Plasmid pFK1 was isolated from transformants producing cellulase, and the cloned cellulase gene was found to be in a 4 X 6 kb HindIII fragment. The cellulase gene was subcloned in a functional state on a 2 X 9 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 800 amino acids. The pFK1-encoded cellulase had the same enzymic properties as the extracellular cellulase produced by the alkalophilic Bacillus sp. strain 1139, but its Mr was slightly higher.     

Characterization of botulinum type A neurotoxin gene: delineation of the N-terminal encoding region

A 456 basepair HindIII fragment that encoded a portion of the type A botulinum neurotoxin gene was cloned into Escherichia coli using a plasmid vector. DNA sequence analysis revealed that this botulinum DNA insert encoded an open reading frame of 35 amino acid residues of which 34 corresponded to the N-terminal residues of botulinum neurotoxin type A.     

The Root of the Universal Tree of Life Inferred from Anciently Duplicated Genes Encoding Components of the Protein-Targeting Machinery

The key protein of the signal recognition particle (termed SRP54 for Eucarya and Ffh for Bacteria) and the protein (termed SRα for Eucarya and Ftsy for bacteria) involved in the recognition and binding of the ribosome SRP nascent polypeptide complex are the products of an ancient gene duplication that appears to predate the divergence of all extant taxa. The paralogy of the genes encoding the two proteins (both of which are GTP triphosphatases) is argued by obvious sequence similarities between the N-terminal half of SRP54(Ffh) and the C-terminal half of SRα(Ftsy). This enables a universal phylogeny based on either protein to be rooted using the second protein as an outgroup. Phylogenetic trees inferred by various methods from an alignment (220 amino acid positions) of the shared SRP54(Ffh) and SRα(Ftsy) regions generate two reciprocally rooted universal trees corresponding to the two genes. The root of both trees is firmly positioned between Bacteria and Archaea/Eucarya, thus providing strong support for the notion (Iwabe et al. 1989; Gogarten et al. 1989) that the first bifurcation in the tree of life separated the lineage leading to Bacteria from a common ancestor to Archaea and Eucarya. None of the gene trees inferred from the two paralogues support a paraphyletic Archaea with the crenarchaeota as a sister group to Eucarya. Received: 19 March 1998 / Accepted: 5 June 1998     

Nucleotide sequence of bglC, the gene specifying enzymeIIbgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product

The EnzymeIIbgl of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of beta-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3.6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in beta-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66.48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2.3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the lambda PL promoter and then transformed into a host strain which produces thermolabile cI857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.     

The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence.

The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking. Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high abundance of methionine residues (M-domain). Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities. Upon proteolysis the intact G-domain was released from SRP, whereas the M-domain remained attached to the core of the particle. Reconstitution experiments demonstrated that the isolated M-domain associates with 7SL RNA in the presence of SRP19. In addition, we observed a specific binding of the M-domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA. This shows that the M-domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA. Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M-domain of SRP54. These results imply that the M-domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G-domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.     

Identification of an RNA-binding domain in human SRP72

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a crucial role during the delivery of secretory proteins from the ribosome to the cell membrane. Among the six proteins of the eukaryotic SRP, the 72 kDa protein (SRP72) is the largest and least characterized. Polypeptides corresponding to various regions of the entire human SRP72 sequence were expressed in Escherichia coli, purified, and partially proteolyzed. Human SRP RNA bound with high affinity to a 63 amino acid residue region near the C terminus of SRP72. Mild treatment of the fragment with chymotrypsin abolished its RNA-binding activity. A conserved sequence with the consensus PDPXRWLPXXER was identified within a 56 amino acid residue RNA-binding domain. Sucrose gradient centrifugation and filter-binding analysis using mutant SRP RNAs showed that SRP72 bound to the moderately conserved portion of SRP RNA helix 5. Nine tetratricopeptide-like repeats (TPRs) poised to interact with other SRP or ribosomal proteins were predicted in the NH2-terminal region. These identifications assign two important functions to a large portion of SRP72 and demonstrate the RNA-binding capacity of the protein.     

The signal sequence interacts with the methionine-rich domain of the 54- kD protein of signal recognition particle

The signal sequence of nascent preprolactin interacts with the 54-kD protein of the signal recognition particle (SRP54). To identify the domain or site on SRP54 that interacts with the signal sequence we used a photocross-linking approach followed by limited proteolysis and immunoprecipitation using anti-peptide antibodies specific for defined regions of SRP54. We found that the previously identified methionine-rich RNA-binding domain of SRP54 (SRP54M domain) also interacts with the signal sequence. The smallest fragment that was found to be crosslinked to the signal sequence comprised the COOH-terminal 6-kD segment of the SRP54M domain. No cross-link to the putative GTP-binding domain of SRP54 (SRP54G domain) was found. Proteolytic cleavage between the SRP54M domain and SRP54G domain did not impair the subsequent interaction between the signal sequence and the SRP54M domain. Our results show that both the RNA binding and signal sequence binding functions of SRP54 are performed by the SRP54M domain.     

An alternative to ITS, a hypervariable, single-copy nuclear intron in corals, and its use in detecting cryptic species within the octocoral genus Carijoa

Here we report a highly variable nuclear marker that can be used for both soft and stony corals. Primers that amplify a ∼177 bp fragment from the nuclear gene encoding the 54 kDa subunit of the signal recognition particle (SRP54) were developed for the octocoral genus Carijoa. Cloning results from 141 individuals suggest that this hypervariable nuclear locus is a single-copy gene. Sequencing revealed a potential cryptic species previously thought to be Carijoa riisei. Results from an Analysis of Molecular Variance (AMOVA) based on mitochondrial DNA (mtDNA) explained <10% of the variation between Atlantic and Pacific samples of C. riisei (F _st = 0.47), whereas the same comparison with SRP54 explained >33% of the variation (F _st = 0.54). Using previously reported degenerate primers for SRP54, high levels of sequence variation were found at this locus across both scleractinian and octocorals. For example, pairwise sequence divergence within octocorals was ∼8–13 times greater with SRP54 than with mtDNA, and, up to 2.8% pairwise sequence divergence was found in SRP54 among individuals of Pocillopora whereas no variation at all was found in mtDNA markers. This case study with the octocoral C. riisei shows that variation in SRP54 appears sufficient to address questions of phylogeography as well as systematics of closely related species.     

Structure of a Bacillus subtilis endo-beta-1,4-glucanase gene.

The nucleotide sequence of the portion of a Bacillus subtilis (strain PAP115) 3 kb Pst I fragment which contains an endo-beta-1, 4-glucanase gene has been determined. This gene encodes a protein of 499 amino acid residues (Mr = 55,234) with a typical B. subtilis signal peptide. Escherichia coli which has been transformed with this gene produces an extracellular endoglucanase with an amino-terminus corresponding to the thirtieth encoded amino acid residue. The gene is preceded by a cryptic reading frame with a rho-independent terminator structure, and itself has such a structure in the immediate 3'-flanking region. We have also identified, in the 5'-flanking region, nucleotide sequences which resemble promoter elements recognized by Bacillus RNA polymerase E sigma 43. Comparison of the encoded amino acid sequence to other known beta-glucanases reveals a small region of similarity to the encoded protein of the Clostridium thermocellum celB gene. These similar regions may contain substrate-binding and/or catalytic sites.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.