2000 Fiscal Year Final Research Report Summary
Studies on the structure and function of A-factor receptor based on its X-ray crystallographic structure
| Project/Area Number |
11306008
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | THE UNIVERSITY OF TOKYO |
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Principal Investigator |
HORINOUCHI Sueharu Graduate School of Agriculture and Life Sciences, THE UNIVERSITY OF TOKYO, Professor, 大学院・農学生命科学研究科, 教授 (80143410)
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| Co-Investigator(Kenkyū-buntansha) |
OHNISHI Yasuo Graduate School of Agriculture and Life Sciences, THE UNIVERSITY OF TOKYO, Assistant Professor, 大学院・農学生命科学研究科, 助手 (90292789)
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| Project Period (FY) |
1999 – 2000
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| Keywords | A-factor / Streptomyces / A-factor receptor / X-ray chrystallography / microbial hormone / secondary metabolism / morphogenesis |
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| Research Abstract |
A chemical signaling molecule A-factor at an extremely low concentration controls secondary metabolism and morphological development in Streptomyces griseus. A rapid method for purification of the A-factor receptor protein was established for crystallization of the receptor. The crystals of the receptor and heavy metal-containing receptor show multiforms, which impeded collection of the difraction data. We are currently collecting data on serenomethione-substituted crystals on Spring 8 at Harima.
More than 20 DNA fragments bound by a transcriptional factor AdpA, one of the targets of the A-factor recepto. Larger DNA fragments covering the isolated fragments were cloned, and their nucleotide sequences were determined. A-factor-dependent transcription of the genes in the cloned fragments was confirmed by S1 nuclease mapping. One of the A-factor-dependent genes was identified as an extracytoplasmic function σ factor of RNA polymerase. This σ factor concerned only with morphological development and not with secondary metabolic function. Additional results are summarized as follows. i) The autophosphorylation of AfsK, a protein serine/threonine kinase, was regulated by means of protein-protein interaction with an AfsK-binding protein KbpA.ii) The whole gene cluster for yellow pigment production, which is under the control of A-factor, was cloed and its nucleotide sequence aws determined. This gene cluster was transcribed by a specific σ^B-like factor. iii) A melanine production genes, rppA and P-450, were cloned. The enzymatic function of RppA was revealed ; it condensated 5 molecules of malonyl-CoA and circulized to form tetrahydroxynaphthalene. Further studied for elucidation of the molecular mechanism of binding and dissociation of A-factor/receptor on the basis of its 3D structure and for revealing each step in the A-factor regulatory cascade will reveal the overall picture of the hormonal control in Streptomyces.
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