Differential regulation of the P1 and P2 promoters of the ribosomal RNA operons in Bacillus subtilis.
Item
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Title
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Differential regulation of the P1 and P2 promoters of the ribosomal RNA operons in Bacillus subtilis.
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Identifier
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AAI9732945
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identifier
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9732945
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Creator
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Liu, David Xiaoming.
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Contributor
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Adviser: Rivka Rudner
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Microbiology
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Abstract
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The expression of ribosomal RNA operons (rrn) of B. subtilis during fast and slow growth, amino acid starvation and carbon source limitation were examined through the use of single-copy spoVG-lacZ fusions integrated at the heterologous amyE. The four rrn promoters tested, rrnO, rrnJ, rrnB, and rrnD, were all growth-rate regulated. Assays of {dollar}\beta{dollar}-galactosidase activity and levels of lacZ mRNA showed that solitary P2 promoters were 2-4 times stronger than the solitary P1 promoters for both rrnO and rrnJ operons. The expression levels also revealed that the promoter upstream region enhances promoter activity. Both P1 and P2 promoters were growth-rate regulated. Only the strong P2 promoters of the rrnO and rrnJ responded to carbon-source limitation. Their activities were decreased by 70% after 90 minutes of {dollar}\alpha{dollar}-methyl glucoside treatment. Both P1 and P2 promoters of rrnO and rrnJ were subject to stringent condition induced by amino acid starvation. After 60 minutes exposure to 2 mg/ml serine hydroxamate, rrnO-P1 and rrnO-P2 both decreased by 85% in activity; rrnJ-P1 and rrnJ-P2 decreased by 60% and 75% in activity, respectively.;The in vitro transcription activity of the rrnO-P1, rrnO-P2, and rrnO-P1P2 with or without UAS region was examined using purified B. subtilis RNA polymerase. The P2 promoters, in solitary P2 or in tandem P1P2 configuration, were highly active and were insensitive to NaCl concentration change. The P1 promoters, however, whether in separated or in tandem P1P2 form, were equally active to P2 only at very low (10-20 mM) NaCl concentrations. Its activity decreased precipitously as NaCl concentration increased, and was undetectable at 200 mM NaCl. In the natural P1P2 configuration, the P2/P1 ratios were about 1-2 at 10-50 mM NaCl, 10-20 at 100-150 mM NaCl. In solitary forms, the P2/P1 ratios were about 3 at 50 mM NaCl and about 7 at 150 mM NaCl.;The fragments of rrnO-UAS, rrnJ-P1, rrnJ-P2, and rrn-P1P2 were found in gel retardation assays capable of forming complexes with protein(s) in the protein extract preparations isolated from B. subtilis strain IS58 or purified E. coli Fis protein. The rrnO-UAS binding protein of B. subtilis is expressed at the early log phase. Although its expression pattern resembles that of E. coli Fis protein, the B. subtilis protein shares no homology with E. coli Fis protein as judged from immunoblot analysis. It is unclear what role the unidentified B. subtilis protein plays in the B. subtilis rRNA gene regulation.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
