| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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.
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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. In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF-G). To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis. Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF-G-GDP from the ribosome. On the other hand , depletion of 4.5S RNA led to the retention of a significant amount of EF-G on 70S ribosomes. In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF-G-binding site at positions 1068-1077 on 23S RNA. We therefore examined by gel mobility-shift assay whether or not mutations in the domain-IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF-G to 23S RNA. Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity. Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF-G with the ribosomes. Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten-base sequence conserved between the two RNAs. Less
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