1988 Fiscal Year Final Research Report Summary
Structure and Function of the RodA Protein Responsible for the Rod Shape of Escherichia coli
| Project/Area Number |
62560072
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | University of Tokyo |
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Principal Investigator |
MATSUZAWA Hiroshi Faculty of Agriculture, University of Tokyo, 農学部, 助教授 (00011966)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Escherichia coli / rod shape of bacteria / cell division / peptidoglycan synthetase / the rodA gene / RodA protein / transmembrane structure of a protein / 遺伝子工学 |
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| Research Abstract |
In this study, the structure and function of the RodA protein of Escherichia coli were analysezed by the methods of protein chemistry and gene engineering.
1. the rodA gene is located 5 nucleotides downstream of the pbpA gene, encoding penicillin-binding protein (PBP) 2. Overproduced RodA protein was purified by SDS-PAGE. The determined N-terminal amino acid sequence showed that the protein has no cleavable signal sequene, but its N-terminal Met residue is removed.
2. To determine the transmembrane structure of the RodA protein, plasmids carrying rodA-bla gene fusions were constructed. Based on the ampicillin resistance of the cells producing RodA-beta-lactamase fusion protein and the hydropathy of the RodA protein at the fused region, localization of the fused point was determined to be in the cytoplasm, cytoplasmic membrane or periplasm. The results obtained showed that the RodA protein traverses the membrane eight times with its N- and C-terminals in the cytoplasm.
3. The ftsL gene is the structural gene for PBP 3. sui mutation was reported, which is a kind of rodA mutation and suppresses a temperature-sensitive lethal mutation of ftsL. The roda(sui) gene was cloned, and its determined nucleotide sequence indicated that the sui mutation is a mutation of CAG (Gln-III) to amber termination codon, TAG, in the rodA gene. Suppressor strain KJBI, which contains both ftsL(Ts) and sui mutations, also has supE mutaion. supE can partially replace the termination signal with Glh. It is considered that the reduction of the RodA protein caused by sui mutaion suppresses the low activity of PBP 3. It is suggested that the RodA protein, probably as a RodA-PBP 2 complex, is involved in the cell division together with PBP 3.
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