|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1992 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Col-plasmid-encoded colicins E3 and E6 have an RNase activity, on their C-terminal T2A domains, which cleaves 16S-RNA to inactivate ribosomes of sensitive E.coli cells. The plasmid protects the host cell from its own colicin action by producing an inhibitor protein, ImmE3 or ImmE6, which binds to T2A.Although E3/ImmE3 and E6/ImmE6 have extensive homology, the protection specificities are completely different between the two sets. A small number of non-identical amino acids (aa) between E3 and E6 sequences are confined in the T2A domains, as well as in the Imm proteins. The project analyzed the specificity-determination of T2A and Imm of E3 and E6, as studied genetically and structural-chemically.
1. The specificity determinants of E3 and E6 toward ImmE3 and ImmE6 were identified through evaluation of stability or Iethality of plasmids which carried chimeric colicin genes between colE3 and colE6 on their expression together with immE3 or immE6. In both T2A and Imm, only one to three aa determine the recognition specificity of the cognate partner protein.
2. The possible catalytic center of E3 were proposed as His513 and Glu517 based on a rational and systematic aa mutation of the T2A domain.
3. The tertiary structure of ImmE3 was determined by multidimensional NMR spectroscopy. The main motif was four-stranded anti-parallel beta-sheet, of which one side is exposed to the solution and contains the side groups of the specificity determinants.
4. Chemical shift perturbation analysis on the HMQC spectra of hemi-labeled complexes, 15N-T2A/14N-Imm and 14N-T2A/15N-Imm, showed the contact area of ImmE6 facing T2A.Interestingly, the specificity determinants of ImmE6 were at the circumference of this area, not at the center.