| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Escherichia coil rnlA was identified as an essential gene for RNase LS. In fact, His-tagged RnlA exhibited an endoribonulcease activity in vitro. However, this activity was too simple and low in comparison with that in vivo. Characterization of RNase LS activity found in a cell extract revealed that this RNase consisted of a large complex, the mass of which was estimated 1000 kDa. In fact, His-RulA was strongly suggested by western blotting to be involved in the complex. We pulled down His-RnlA with Ni-beads to find proteins associated with His-RnlA and twelve RnlA-associating proteins including TpiA and SucB were determined by aminoacid sequencing. Because the RNase LS activity was impaired by disruption of tpiA or sucB. we conclude that the 1000-kDa complex is an integral part of RNase LS. rnlB locates immediately downstream of rnlA in the E. coli genomic map. When we disrupted rnlB, the mutant exhibited no RNase LS activity. In addition, pull-down of His-RnlB from cell extract also
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precipitated RnlA by Ni-beads, and these proteins were coeluated from the beads with imidazole. These results strongly suggests that RnlB is also involved in the 1000-kDa complex.
RNase LS was formerly suggested to cleave mRNA depending translational ability of the mRNA. In this research, we established that RNase LS cleaves mRNA downstream of a termination codon depending on a translation termination process.
We found that immediately after T4 infection, RNases E and G are activated to degrade host mRNAs rapidly. This degradation required the expression of T4 genes, because such rapid degradation did not occurr in the presence of rifampicin added in prior to T4 infection. In order to search for the causal T4 genes, we tested several deletion mutants of T4 and found that the causal genes are involved in TK2 region. Since TK2 region contains 20 unknown genes, we divided this region into 4 sub-regions and prepared deletion mutants that lacked each sub-region or a combination of sub-regions. Using the deletin mutants, we found that activation of RNase E required one of the sub-regions and that activation of RNase G required two of four sub-regions. Thus, RNases E and G are activated by a different set of T4 genes. Less
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