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1.
The archaeal protein L7Ae and eukaryotic homologs such as L30e and 15.5kD comprise the best characterized family of K-turn-binding proteins. K-turns are an RNA motif comprised of a bulge flanked by canonical and noncanonical helices. They are widespread in cellular RNAs, including bacterial gene-regulatory RNAs such as the c-di-GMP-II, lysine, and SAM-I riboswitches, and the T-box. The existence in bacteria of K-turn-binding proteins of the L7Ae family has not been proven, although two hypothetical proteins, YbxF and YlxQ, have been proposed to be L7Ae homologs based on sequence conservation. Using purified, recombinant proteins, we show that Bacillus subtilis YbxF and YlxQ bind K-turns (K(d) ~270 nM and ~2300 nM, respectively). Crystallographic structure determination demonstrates that both YbxF and YlxQ adopt the same overall fold as L7Ae. Unlike the latter, neither bacterial protein recognizes K-loops, a structural motif that lacks the canonical helix of the K-turn. This property is shared between the bacterial and eukaryal family members. Comparison of our structure of YbxF in complex with the K-turn of the SAM-I riboswitch and previously determined structures of archaeal and eukaryal homologs bound to RNA indicates that L7Ae approaches the K-turn at a unique angle, which results in a considerably larger RNA-protein interface dominated by interactions with the noncanonical helix of the K-turn. Thus, the inability of the bacterial and eukaryal L7Ae homologs to bind K-loops probably results from their reliance on interactions with the canonical helix. The biological functions of YbxF and YlxQ remain to be determined. 相似文献
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Mechanism of erythromycin-induced ermC mRNA stability in Bacillus subtilis. 总被引:10,自引:5,他引:5
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In Bacillus subtilis, the ermC gene encodes an mRNA that is unusually stable (40-min half-life) in the presence of erythromycin, an inducer of ermC gene expression. A requirement for this induced mRNA stability is a ribosome stalled in the ermC leader region. This property of ermC mRNA was used to study the decay of mRNA in B. subtilis. Using constructs in which the ribosome stall site was internal rather than at the 5' end of the message, we show that ribosome stalling provides stability to sequences downstream but not upstream of the ribosome stall site. Our results indicate that ermC mRNA is degraded by a ribonucleolytic activity that begins at the 5' end and degrades the message in a 5'-to-3' direction. 相似文献
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Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase. 总被引:5,自引:1,他引:4
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We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion. 相似文献
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The construction of plasmid pVKH300, which is useful for modifying any promoter into the thermoregulated form in B. subtilis cells, is presented. The main features of the plasmid are the presence of effectively expressed in B. subtilis lambda C1857 gene and recognition site of BglII restriction enzyme between OR2 and OR3 lambda phage operator sites. Promoterless alpha-amylase gene of B. amyloliquefaciens is used as a reporter gene for promoter cloning into BglII site of pVKH300. Examples of promoter-containing DNA fragments cloning with pVKH300 as vector are presented. It was found that the best regulated promoter, in a plasmid named pVKH332, was cloned in such a way that the distance between central nucleotides of OR2 and OR3 is equal to integer number of DNA helix turns (84 b.p. in the case). 相似文献
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Bélanger F Gagnon MG Steinberg SV Cunningham PR Brakier-Gingras L 《Journal of molecular biology》2004,338(4):683-693
The 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) is amongst the most conserved regions of rRNA. This tetraloop forms a GNRA motif that docks into the minor groove of three base-pairs at the bottom of helix 24 of 16S rRNA in the 30S subunit. Both the tetraloop and its receptor in helix 24 contact the 23S rRNA, forming the intersubunit bridge B2c. Here, we investigated the interaction between the 900 tetraloop and its receptor by genetic complementation. We used a specialized ribosome system in combination with an in vivo instant evolution approach to select mutations in helix 24 compensating for a mutation in the 900 tetraloop (A900G) that severely decreases ribosomal activity, impairing subunit association and translational fidelity. We selected two mutants where the G769-C810 base-pair of helix 24 was substituted with either U-A or C x A. When these mutations in helix 24 were investigated in the context of a wild-type 900 tetraloop, the C x A but not the U-A mutation severely impaired ribosome activity, interfering with subunit association and decreasing translational fidelity. In the presence of the A900G mutation, both mutations in helix 24 increased the ribosome activity to the same extent. Subunit association and translational fidelity were increased to the same level. Computer modeling was used to analyze the effect of the mutations in helix 24 on the interaction between the tetraloop and its receptor. This study demonstrates the functional importance of the interaction between the 900 tetraloop and helix 24. 相似文献
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Nucleotide sequence of the gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex. 总被引:11,自引:8,他引:3
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The nucleotide sequence was determined for the first part of the Bacillus subtilis sdh operon. An open reading frame corresponding to the structural gene, sdhA, for cytochrome b558 was identified. The predicted molecular weight of the cytochrome (excluding the N-terminal methionine) is 22,770. It is a very hydrophobic protein with five probable membrane-spanning segments. There is little homology between the B. subtilis cytochrome b558 and cytochrome b of mitochondrial complex III from different organisms or between cytochrome b558 and the hydrophobic sdhC and sdhD peptides of the Escherichia coli sdh operon. About 30 bases downstream of the sdhA stop codon, a new open reading frame starts. The nucleotide sequence predicts the presence of a typical flavin-binding peptide which identifies this reading frame as part of the sdhB gene. Seven bases upstream of the sdhA initiation codon ATG there is a typical B. subtilis ribosome binding site (free energy of interaction, -63 kJ), and further upstream, tentative sigma 55 and sigma 32 promoter sequences were found. The upstream region also contains two 12-base-long direct repeats; their significance is unknown. 相似文献
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New plasmid expression vectors for Bacillus subtilis 总被引:2,自引:0,他引:2
The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells. 相似文献
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A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome 总被引:1,自引:0,他引:1
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Bélanger F Théberge-Julien G Cunningham PR Brakier-Gingras L 《RNA (New York, N.Y.)》2005,11(6):906-913
The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA. 相似文献
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Expression of the MuIFN alpha 7 gene in Bacillus subtilis using the levansucrase system 总被引:1,自引:0,他引:1
The mouse interferon alpha 7 gene, the signal sequence of which has been removed by oligonucleotide-directed mutagenesis, was introduced into a Bacillus subtilis secretion vector containing the promoter and the signal sequence of the B. subtilis levansucrase gene. Different B. subtilis strains were transformed with the fused levansucrase-interferon gene; their cell extracts and culture supernatants tested for antiviral activity and the IFN alpha 7 protein showed the presence of IFN alpha 7 only in the cell extracts. To promote IFN alpha 7 secretion, constructs were realized in order to restore the alpha helix conformation of the signal sequence of levansucrase and interferon protein junction. Our results suggest that factors other than the structure of the peptide around the cleavage site are involved in the secretion of IFN alpha 7 by B. subtilis. 相似文献
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The staphylococcal nuclease (nuc) gene from Staphylococcus aureus has been cloned and expressed in Bacillus subtilis. The nuclease protein was expressed either from its own promoter and translation start signals, or from a combination of a B. subtilis promoter, ribosome binding site, and a signal peptide sequence. Greater than 80% of the active gene product was secreted into the medium, whereas, when a signal peptide sequence was absent, as little as 4% of the nuclease activity was found in the culture medium. Intracellular (or cell-bound) nuclease, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, was shown to have the molecular weight of the predicted precursor protein with the signal peptide. Levels of nuclease reached 50 mg per liter in the culture medium, depending on the growth medium and the strain used. These findings indicate the prospective use of nuclease as a model system for studying secretion of heterologous proteins in B. subtilis. 相似文献
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Byung-Sik Shin Joo-Ran Kim Michael G. Acker Kathryn N. Maher Jon R. Lorsch Thomas E. Dever 《Molecular and cellular biology》2009,29(3):808-821
The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit. 相似文献
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SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem–loop in the 3′ UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA. Diepoxybutane cross-linking confirmed the interaction of SBP2 with helix ES7L-E. Additionally, binding of SBP2 to the ribosome led to increased reactivity toward chemical probes of a few bases in ES7L-E and in the universally conserved helix H89, indicative of conformational changes in the 28S rRNA in response to SBP2 binding. This study revealed for the first time that SBP2 makes direct contacts with a discrete region of the human 28S rRNA. 相似文献
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Trans-translation, in which a ribosome switches between translation of an mRNA and a tmRNA, produces a chimera polypeptide of an N-terminal truncated polypeptide and a C-terminal tag-peptide encoded by tmRNA. One of the tmRNA binding proteins, a ribosomal protein S1, has not been found in a group of Gram-positive bacteria. In this study, the trans-translation reaction with tmRNA from Bacillus subtilis belonging to this group was examined. When a truncated gene lacking a termination codon was expressed in B. subtilis, a 15-amino acid tag-peptide derived from tmRNA was identified in the C-termini of the trans-translation products. An identical tag-peptide was also found at the C-termini of the products from a truncated gene, when it was coexpressed with B. subtilis tmRNA in Escherichia coli. B. subtilis tmRNA was functional, although much less efficiently, in the in vitro poly(U)-dependent tag-peptide synthesis system of E. coli. A comparison of two bacterial tmRNAs suggests that the rule for determining the tag-initiation point on tmRNA may be the same in Gram-positive and Gram-negative bacteria. 相似文献
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Intersubunit bridges are important for holding together subunits in the 70S ribosome. Moreover, a number of intersubunit bridges have a role in modulating the activity of the ribosome during translation. Ribosomal intersubunit bridge B2a is formed by the interaction between the conserved 23S rRNA helix-loop 69 (H69) and the top of the 16S rRNA helix 44. Within the 70S ribosome, bridge B2a contacts translation factors and the A-site tRNA. In addition to bridging the subunits, bridge B2a has been invoked in a number of other ribosomal functions from initiation to termination. In the present work, single-nucleotide substitutions were inserted at positions 1912 and 1919 of Escherichia coli 23S rRNA (helix 69), which are involved in important intrahelical and intersubunit tertiary interactions in bridge B2a. The resulting ribosomes had a severely reduced activity in a cell-free translation elongation assay, but displayed a nearly wild-type-level peptidyl transferase activity. In vitro reassociation efficiency decreased with all of the H69 variant 50S subunits, but was severest with the A1919C and ΔH69 variants. The mutations strongly affected initiation-factor-dependent 70S initiation complex formation, but exhibited a minor effect on the nonenzymatic initiation process. The mutations decreased ribosomal processivity in vitro and caused a progressive depletion of 50S subunits in polysomal fractions in vivo. Mutations at position 1919 decreased the stability of a dipeptidyl-tRNA in the A-site, whereas the binding of the dipeptidyl-tRNA was rendered more stable with 1912 and ΔH69 mutations. Our results suggest that the H69 of 23S rRNA functions as a control element during enzymatic steps of translation. 相似文献
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There are two major domains of interaction between the Escherichia coli release factors (RF-1 and RF-2) and each subunit of the ribosome. RF-2 has a binding domain on the shoulder and lower head region of the small subunit at the small lobe distant from the decoding site. This is in close proximity to one of the domains on the large subunit which includes the body dimer of L7/L12 and L11. The other domains of interaction, at the decoding site on the small subunit, and at the peptidyltransferase centre of the large subunit of the ribosome, are some distance from the first two, although the evidence for direct contact with the ribosome is less comprehensive. The release factors may therefore have two distinct structural domains, and in support of this concept RF-1 and RF-2 can both be cleaved into two fragments by papain. Region-specific antibodies, and antibodies against defined peptide within the RF sequences have given an indication that a significant part of an interacting RF molecule is in close proximity to the ribosome surface, confirming an observation by immunoelectron microscopy which suggested that the RF penetrates deeply into the cleft between the two subunits. A region of highly conserved primary sequence between the two release factors from E coli is also conserved in those from B subtilis suggesting it forms an important structural or functional domain. Antibodies against peptides from the N-terminal end of this region strongly inhibit binding of the RF to the ribosome. 相似文献