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1.
A segment of early RNA from Bacillus subtilis bacteriophage SP82 was shown to function as a 5' stabilizer in B. subtilis. Several heterologous RNA sequences were stabilized by the presence of the SP82 sequence at the 5' end, and expression of downstream coding sequences was increased severalfold. The SP82 RNA segment encodes a B. subtilis RNase III cleavage site, but cleavage by B. subtilis RNase III was not required for stabilization. The sequence that specifies 5' stabilizer function was localized to a polypurine sequence that resembles a ribosome binding site. The ability of the SP82 sequence to stabilize downstream RNA was dependent on its position relative to the 5' end of the RNA. These results demonstrate the existence of a new type of 5' stabilizer in B. subtilis and indicate that attack at the 5' end is a principal mechanism for initiation of mRNA decay in B. subtilis.  相似文献   

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An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.  相似文献   

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A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al ., 1996 — accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase). Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B. subtilis and Escherichia coli PNPase enzymes.  相似文献   

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Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.  相似文献   

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The Bacillus subtilis RNA polymerase sigma 43 subunit and the phage SP82 encoded 28-kDa peptide are responsible for the binding of RNA polymerase to early and middle SP82 promoters, respectively. The delta peptide enhances the specificity of the interaction of B. subtilis RNA polymerase with these promoters. We have used sedimentation experiments to determine the effect of each of the three specificity factors, delta, sigma, and the 28-kDa peptide, on the binding of the other two factors to RNA polymerase core and the effect of NaCl on these binding equilibria. We show that sigma 43 and the 28-kDa peptide can each bind to RNA polymerase core at the same time as delta. Sigma 43 and the 28-kDa peptide have similar affinities to core at 0.1 M NaCl, but the 28-kDa peptide binds to core-delta more strongly than sigma 43. The implications of these findings with respect to the replacement of sigma 43 by the 28-kDa peptide and the mechanism of promoter search by B. subtilis RNA polymerase are discussed.  相似文献   

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Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell.  相似文献   

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Secondary structure of splice sites in adenovirus mRNA precursors.   总被引:7,自引:4,他引:3       下载免费PDF全文
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F Braun  J Le Derout    P Régnier 《The EMBO journal》1998,17(16):4790-4797
The hypothesis generally proposed to explain the stabilizing effect of translation on many bacterial mRNAs is that ribosomes mask endoribonuclease sites which control the mRNA decay rate. We present the first demonstration that ribosomes interfere with a particular RNase E processing event responsible for mRNA decay. These experiments used an rpsO mRNA deleted of the translational operator where ribosomal protein S15 autoregulates its synthesis. We demonstrate that ribosomes inhibit the RNase E cleavage, 10 nucleotides downstream of the rpsO coding sequence, responsible for triggering the exonucleolytic decay of the message mediated by polynucleotide phosphorylase. Early termination codons and insertions which increase the length of ribosome-free mRNA between the UAA termination codon and this RNase E site destabilize the translated mRNA and facilitate RNase E cleavage, suggesting that ribosomes sterically inhibit RNase E access to the processing site. Accordingly, a mutation which reduces the distance between these two sites stabilizes the mRNA. Moreover, an experiment showing that a 10 nucleotide insertion which destabilizes the untranslated mRNA does not affect mRNA stability when it is inserted in the coding sequence of a translated mRNA demonstrates that ribosomes can mask an RNA feature, 10-20 nucleotides upstream of the processing site, which contributes to the RNase E cleavage efficiency.  相似文献   

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Three observations indicated that the 2-megadalton chloramphenicol resistance plasmid pCM194 interferes with SP02 lysogeny of Bacillus subtilis. SP02 plaques formed on B. subtilis(pCM194) appeared almost clear, whereas plaques produced on plasmid-free or pUB110-containing cells contained large turbid centers. The number of phages spontaneously liberated by B. subtilis(SP02) was increased 10-fold or more when pCM194 was also present in the lysogens. Lastly, growth of B. subtilis(SP02, pCM194) for approximately 20 to 25 generations resulted in essentially complete loss of the prophage. This interference was not observed with pUB110 or pE194, and the pCM194 interference was not directed against B. subtilis temperate phage phi 105, which is unrelated to SP02. Lytic replication of SP02 appeared to be unaffected by pCM194. pCM194 interference with SP02 lysogeny was demonstrable in recombination-proficient strains and a recE mutant of B. subtilis. SP02 prophage which were noninducible due to the phage ind mutation were resistant to pCM194 interference. pCM194 interference was lost when the entire pCM194 molecule was joined at its unique HpaII site or at one of the two MboI sites to pUB110 or pUB110 derivatives. pBR322 joined to pCM194 at the same MboI site or at the HindIII site produced chimeras that retained the ability to interfere with SP02 lysogeny. A three-part plasmid constructed by joining pBR322 to pCM194 (at HindIII sites) and to pE194 (at PstI sites) was compatible with the SP02 prophage and showed a temperature-sensitive replication phenotype characteristic of the pE194 replicon. One explanation for the interference involves competition for a host component between an SP02 genome attempting to establish lysogeny and plasmids whose replication is directed by the pCM194 replicon.  相似文献   

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The 3'-end of the RNA genome of turnip yellow mosaic virus can form a pseudoknotted tRNA-like structure that can be recognized by several tRNA-specific enzymes. We have found that the catalytic RNA component of Bacillus subtilis RNase P can cleave this structure in unusually low ionic strength buffers at a site analogous to the 5'-end of an aminoacyl stem of a tRNA. Most other precursors can only be processed under low ionic strength conditions if the RNase P holoenzyme is used; processing by the catalytic RNA component alone requires a higher ionic strength buffer. The cleavage of the turnip yellow mosaic virus tRNA-like structure demonstrates the importance of the substrate in determining the optimal buffer conditions for this reaction and also shows that high ionic strength buffers are not always necessary for cleavage by the catalytic RNA.  相似文献   

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