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Kunio Yamane - One of the best experts on this subject based on the ideXlab platform.
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bacillus subtilis histone like protein hbsu is an integral component of a srp like particle that can bind the alu domain of Small Cytoplasmic RNA
Journal of Biological Chemistry, 1999Co-Authors: Kouji Nakamura, Takao Yamazaki, Shouichi Yahagi, Kunio YamaneAbstract:Small Cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a teRNAry complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.
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depletion of bacillus subtilis histone like protein hbsu causes defective protein translocation and induces upregulation of Small Cytoplasmic RNA
Biochemical and Biophysical Research Communications, 1999Co-Authors: Takao Yamazaki, Kouji Nakamura, Shouichi Yahagi, Kunio YamaneAbstract:Small Cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.
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bacillus subtilis RNAse iii cleaves both 5 and 3 sites of the Small Cytoplasmic RNA precursor
Journal of Biological Chemistry, 1998Co-Authors: Akihiro Oguro, Hiroshi Kakeshita, Kouji Nakamura, Kunio Yamane, Wei Wang, David H BechhoferAbstract:Abstract Bacillus subtilis Small Cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5′- and 3′-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNAse III. The B. subtilis enzyme that has similar activity toEscherichia coli RNAse III has been purified and is designated Bs-RNAse III. Recently, B. subtilis rncS has been shown to encode Bs-RNAse III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379–7385). We show here that Bs-RNAse III and the purified His-tagged product of rncScleave pre-scRNA at both 5′- and 3′-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3′-site is less efficient. The 5′-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3′-end. Bs-RNAse III cleavage yields a two-base 3′-overhang, which is consistent with the manner in which E. coli RNAse III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNAse III is an enzyme that processes pre-scRNA.
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identification of a region of bacillus subtilis ffh a homologue of mammalian srp54 protein that is essential for binding to Small Cytoplasmic RNA
Journal of Biological Chemistry, 1996Co-Authors: Kei Kurita, Kouji Nakamura, Kiyofumi Honda, Satoru Suzuma, Hiromu Takamatsu, Kunio YamaneAbstract: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. The amino acid sequence of this region is well conserved among members of the SRP54 protein family. This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407. Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not. The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh. The two glycine residues of the 405GSG407 were also essential. MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not. The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.
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Small Cytoplasmic RNA scRNA gene from clostridium perfringens can replace the gene for the bacillus subtilis scRNA in both growth and sporulation
Microbiology, 1995Co-Authors: Kouji Nakamura, Erika Hashizume, Toshinori Shibata, Yoshio Nakamura, Sarka Mala, Kunio YamaneAbstract:Summary: Small Cytoplasmic RNA (scRNA) is a member of an evolutionary conserved signal-recognition-particle-like RNA family. Using a DNA fragment of Bacillus subtilis scRNA gene as a probe, we cloned and characterized a Clostridium perfringens gene encoding the scRNA. Mapping the 5′ and 3′ ends of scRNA revealed that C. perfringens scRNA consists of 269 nucleotides: the sequence has about 70% primary sequence homology with B. subtilis scRNA. The predicted secondary structure appeared to be similar to that of B. subtilis scRNA, indicating that there are domains I and II in C. perfringens scRNA, in addition to domain IV. Functional analysis showed that C. perfringens scRNA could compensate for vegetative growth and allow the formation of heat-resistant spores in an scRNA-depleted B. subtilis strain, whereas Escherichia coli 4·5S RNA could not maintain sporulation. Since both E. coli 4·5S RNA and C. perfringens scRNA have the same binding specificity to B. subtilis Ffh protein, the difference in complementation activity reflects the function of domains I and II.
Kouji Nakamura - One of the best experts on this subject based on the ideXlab platform.
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bacillus subtilis histone like protein hbsu is an integral component of a srp like particle that can bind the alu domain of Small Cytoplasmic RNA
Journal of Biological Chemistry, 1999Co-Authors: Kouji Nakamura, Takao Yamazaki, Shouichi Yahagi, Kunio YamaneAbstract:Small Cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a teRNAry complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.
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depletion of bacillus subtilis histone like protein hbsu causes defective protein translocation and induces upregulation of Small Cytoplasmic RNA
Biochemical and Biophysical Research Communications, 1999Co-Authors: Takao Yamazaki, Kouji Nakamura, Shouichi Yahagi, Kunio YamaneAbstract:Small Cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.
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bacillus subtilis RNAse iii cleaves both 5 and 3 sites of the Small Cytoplasmic RNA precursor
Journal of Biological Chemistry, 1998Co-Authors: Akihiro Oguro, Hiroshi Kakeshita, Kouji Nakamura, Kunio Yamane, Wei Wang, David H BechhoferAbstract:Abstract Bacillus subtilis Small Cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5′- and 3′-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNAse III. The B. subtilis enzyme that has similar activity toEscherichia coli RNAse III has been purified and is designated Bs-RNAse III. Recently, B. subtilis rncS has been shown to encode Bs-RNAse III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379–7385). We show here that Bs-RNAse III and the purified His-tagged product of rncScleave pre-scRNA at both 5′- and 3′-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3′-site is less efficient. The 5′-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3′-end. Bs-RNAse III cleavage yields a two-base 3′-overhang, which is consistent with the manner in which E. coli RNAse III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNAse III is an enzyme that processes pre-scRNA.
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identification of a region of bacillus subtilis ffh a homologue of mammalian srp54 protein that is essential for binding to Small Cytoplasmic RNA
Journal of Biological Chemistry, 1996Co-Authors: Kei Kurita, Kouji Nakamura, Kiyofumi Honda, Satoru Suzuma, Hiromu Takamatsu, Kunio YamaneAbstract: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. The amino acid sequence of this region is well conserved among members of the SRP54 protein family. This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407. Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not. The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh. The two glycine residues of the 405GSG407 were also essential. MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not. The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.
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Small Cytoplasmic RNA scRNA gene from clostridium perfringens can replace the gene for the bacillus subtilis scRNA in both growth and sporulation
Microbiology, 1995Co-Authors: Kouji Nakamura, Erika Hashizume, Toshinori Shibata, Yoshio Nakamura, Sarka Mala, Kunio YamaneAbstract:Summary: Small Cytoplasmic RNA (scRNA) is a member of an evolutionary conserved signal-recognition-particle-like RNA family. Using a DNA fragment of Bacillus subtilis scRNA gene as a probe, we cloned and characterized a Clostridium perfringens gene encoding the scRNA. Mapping the 5′ and 3′ ends of scRNA revealed that C. perfringens scRNA consists of 269 nucleotides: the sequence has about 70% primary sequence homology with B. subtilis scRNA. The predicted secondary structure appeared to be similar to that of B. subtilis scRNA, indicating that there are domains I and II in C. perfringens scRNA, in addition to domain IV. Functional analysis showed that C. perfringens scRNA could compensate for vegetative growth and allow the formation of heat-resistant spores in an scRNA-depleted B. subtilis strain, whereas Escherichia coli 4·5S RNA could not maintain sporulation. Since both E. coli 4·5S RNA and C. perfringens scRNA have the same binding specificity to B. subtilis Ffh protein, the difference in complementation activity reflects the function of domains I and II.
Jurgen Brosius - One of the best experts on this subject based on the ideXlab platform.
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expression of dendritic bc200 RNA component of a 11 4s ribonucleoprotein particle is conserved in monkey brain
Neuroscience Letters, 1997Co-Authors: Jrgang Cheng, Henri Tiedge, Jurgen BrosiusAbstract:Abstract BC200 RNA, a brain-specific Small Cytoplasmic RNA, is one of the few known cell type specific non-messenger RNAs. It originated from a monomeric Alu short interspersed repetitive element (SINE) in primates. In situ hybridization using rhesus monkey ( Macaca mulatta ) brain sections reveals a similar cellular and sub-cellular distribution as in human brain. In addition to confirming its dendritic location, the distribution in an old world monkey indicates a discrete regional and subcellular location of BC200 RNA. We also report that BC200 RNA exists as a ribonucleoprotein (RNP) particle in vivo. In sucrose gradients, the BC200 particle has a sedimentation constant of about 11.4 S, significantly more than the corresponding 200 nucleotide long naked RNA (~7.6 S).
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bc200 RNA a neural RNA polymerase iii product encoded by a monomeric alu element
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: John A Martignetti, Jurgen BrosiusAbstract:Abstract We demonstrate that the BC200 RNA gene, which encodes a neural Small Cytoplasmic RNA, is a member of the most prodigious family of interspersed repetitive DNA and that its product represents an example of a primate tissue-specific RNA polymerase III transcript. The BC200 RNA gene is an early monomeric member and one of the few postulated transcriptionally active Alu sequences in this family of nearly half a million retropositionally amplified elements dispersed throughout the human genome. Furthermore, the isolation of two pseudogenes, BC200 beta and BC200 gamma, demonstrates the gene's transpositional ability. Interestingly, the BC200 beta pseudogene may have been generated by a conversion-like event after the human/chimpanzee divergence, resulting in an exchange of the left arm of a dimeric Alu element with the BC200 RNA coding sequence. Our data on conserved features of the active BC200 alpha gene suggest that its RNA product has been "exapted" into a function of the primate brain and provides a selective advantage to the species.
Takao Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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bacillus subtilis histone like protein hbsu is an integral component of a srp like particle that can bind the alu domain of Small Cytoplasmic RNA
Journal of Biological Chemistry, 1999Co-Authors: Kouji Nakamura, Takao Yamazaki, Shouichi Yahagi, Kunio YamaneAbstract:Small Cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a teRNAry complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.
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depletion of bacillus subtilis histone like protein hbsu causes defective protein translocation and induces upregulation of Small Cytoplasmic RNA
Biochemical and Biophysical Research Communications, 1999Co-Authors: Takao Yamazaki, Kouji Nakamura, Shouichi Yahagi, Kunio YamaneAbstract:Small Cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.
David H Bechhofer - One of the best experts on this subject based on the ideXlab platform.
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processing of bacillus subtilis Small Cytoplasmic RNA evidence for an additional endonuclease cleavage site
Nucleic Acids Research, 2007Co-Authors: Shiyi Yao, Joshua B Blaustein, David H BechhoferAbstract:Small Cytoplasmic RNA (scRNA) of Bacillus subtilis is the RNA component of the signal recognition particle. scRNA is transcribed as a 354-nt precursor, which is processed to the mature 271-nt scRNA. Previous work demonstrated the involvement of the RNAse III-like endoribonuclease, Bs-RNAse III, in scRNA processing. Bs-RNAse III was found to cleave precursor scRNA at two sites (the 5' and 3' cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3' exoribonuclease to the mature scRNA. Here we show that Bs-RNAse III cleaves primarily at the 5' cleavage site and inefficiently at the 3' site. RNAse J1 is responsible for much of the cleavage that releases scRNA from downstream sequences. The subsequent exonucleolytic processing is carried out largely by RNAse PH.
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bacillus subtilis RNAse iii cleaves both 5 and 3 sites of the Small Cytoplasmic RNA precursor
Journal of Biological Chemistry, 1998Co-Authors: Akihiro Oguro, Hiroshi Kakeshita, Kouji Nakamura, Kunio Yamane, Wei Wang, David H BechhoferAbstract:Abstract Bacillus subtilis Small Cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5′- and 3′-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNAse III. The B. subtilis enzyme that has similar activity toEscherichia coli RNAse III has been purified and is designated Bs-RNAse III. Recently, B. subtilis rncS has been shown to encode Bs-RNAse III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379–7385). We show here that Bs-RNAse III and the purified His-tagged product of rncScleave pre-scRNA at both 5′- and 3′-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3′-site is less efficient. The 5′-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3′-end. Bs-RNAse III cleavage yields a two-base 3′-overhang, which is consistent with the manner in which E. coli RNAse III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNAse III is an enzyme that processes pre-scRNA.