Regulation of the early stage of the tetrapyrrole biosynthetic pathway in Escherichia coli.
Item
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Title
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Regulation of the early stage of the tetrapyrrole biosynthetic pathway in Escherichia coli.
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Identifier
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AAI9807942
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identifier
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9807942
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Creator
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Hua, Wei.
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Contributor
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Mentors: Sharon Cosloy | Charlotte Russell
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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, Genetics | Biology, Microbiology
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Abstract
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Glutamyl-tRNA reductase (GTR), the gene product of hemA, is probably the major site of regulation of ALA synthesis in E. coli. A study was undertaken to understand the regulation of this gene.;There are three putative {dollar}\sigma\sp{lcub}70{rcub}{dollar} promoters (P1, P2 and P3), and one {dollar}\sigma\sp{lcub}32{rcub}{dollar} promoter (P32) in the 5{dollar}\sp\prime{dollar} upstream region of hemA. There are also binding sites for FNR, CRP, NarL and ArcA regulatory proteins, and two overlapping stem loops that resemble the tRNA{dollar}\rm\sp{lcub}Glu{rcub}{dollar} stem loop. Six fragments of this region were constructed and cloned in front of a promoterless gene for CAT on the vector pKK232-8. The promoter strength of these fragments was measured by the CAT ELISA assay. The promoter strength decreased with the size of the fragment in the order: 515 bp {dollar}>{dollar} 359 bp {dollar}>{dollar} 239 bp. Our results indicate that P1 is necessary but not sufficient for hemA expression; that 138 bp upstream of P1 are essential for high level of expression, and that P2 and P3 are weak promoters.;The role of FNR, CRP, NarL and ArcA in hemA regulation was examined by using the above plasmids in strains which harbored mutation in the genes for the transcription factors and in their respective isogenic parents. ArcA stimulated CAT expression about 2-fold during aerobic growth. FNR decreased expression about 1.5-fold. These results correlate with the finding that CAT expression with all of these plasmids was higher in cells grown aerobically as compared to cells grown anaerobically. Aerobic growth on glucose caused a 2-fold decrease in CAT expression in stationary phase but not in log phase cells, and this effect was mediated by CRP. There was no evidence that NarL is involved in the regulation.;Growth of strains harboring the plasmids on rich medium gave higher CAT expression than growth on minimal medium. Starvation for ALA or heme increased CAT expression more than 2-fold.;The levels of inhibition or enhancement of CAT expression driven by hemA promoters are not dramatic. Thus control of hemA expression by regulation of the initiation of transcription may slightly modulate the ability of an E. coli cell to synthesize heme in different environments.
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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.
