Kobiler Oren, Koby Simi, Teff Dinah, Court Donald, Oppenheim Amos B
Department of Molecular Genetics and Biotechnology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14964-9. doi: 10.1073/pnas.222172499. Epub 2002 Oct 23.
ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the lambda CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the lambda CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage lambda carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein.
依赖ATP的蛋白酶，如FtsH（HflB），可识别特定的蛋白质底物。其中之一是λ CII蛋白，它在噬菌体裂解-溶原化决定中起关键作用。在此，我们提供证据表明，CII保守的C末端作为快速蛋白水解的必要且充分的顺式作用靶点。删除该保守标签或在其C末端赋予两个天冬氨酸残基的突变不影响CII的结构或活性。然而，这些突变消除了FtsH对CII的降解作用。我们建立了一种针对λ CIII蛋白的体外检测方法，并证明CIII直接抑制FtsH的蛋白水解作用，以保护CII和CII突变体不被降解。在CII C末端携带突变的噬菌体λ显示溶原化频率增加，这表明CII的这一片段本身可能对影响裂解-溶原化发展的调控敏感。此外，编码CII C末端的区域与一个编码名为OOP的小反义RNA的基因重叠。我们表明，删除cII基因的末端可以阻止反式提供的OOP RNA干扰CII活性。这些发现提供了一个基因的例子，该基因携带一个在mRNA和蛋白质水平调节稳定性的区域。
