Regulation of the gua operon of Escherichia coli by the DnaA protein
Regulation of the gua operon of Escherichia coli by the DnaA protein
The guaBA operon of E.coli determines the production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during nucleotide biosynthesis. Two DnaA boxes, binding sites for the DnaA replication-initiating DnaA protein, are present in the gua operon. One box (with a 8/9 fit to the consensus sequence DnaA box TTATACCAACA) overlaps the gua promoter, the other box (matching the consensus sequence) lies 200 base pairs within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains β-galactosidase acts as a reporter enzyme for transcription initiation at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with a multicopy oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Repression of guaP by DnaA was dependent on the presence of both DnaA boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA. The DnaA protein was purified to near homogeneity from an overproducing strain and supported DNA synthesis in vitro in a complementation assay. Using the techniques of filter-binding and gel retardation, the purified DnaA protein was found to bind restriction fragments from the gua operon. Footprinting demonstrated that DnaA protein binds specifically at the consensus sequence DnaA box within guaB. This box may therefore function as a transcriptional terminator. A computer search of nucleotide sequences in the EMBL data library (release 30, 1992) for the E.coli chromosome revealed 48 sites conforming to the consensus sequence for the DnaA box. These sites are located non-randomly on the chromosome map with a bias towards the origin of replication (oriC). A model is proposed whereby genes containing DnaA boxes (including guaB) comprise a DnaA regulon. Transcriptional activity of these genes may be regulated by the cellular concentration of the DnaA protein.
University of Southampton
1992
Tesfa-Selase, Fisehaye
(1992)
Regulation of the gua operon of Escherichia coli by the DnaA protein.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The guaBA operon of E.coli determines the production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during nucleotide biosynthesis. Two DnaA boxes, binding sites for the DnaA replication-initiating DnaA protein, are present in the gua operon. One box (with a 8/9 fit to the consensus sequence DnaA box TTATACCAACA) overlaps the gua promoter, the other box (matching the consensus sequence) lies 200 base pairs within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains β-galactosidase acts as a reporter enzyme for transcription initiation at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with a multicopy oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Repression of guaP by DnaA was dependent on the presence of both DnaA boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA. The DnaA protein was purified to near homogeneity from an overproducing strain and supported DNA synthesis in vitro in a complementation assay. Using the techniques of filter-binding and gel retardation, the purified DnaA protein was found to bind restriction fragments from the gua operon. Footprinting demonstrated that DnaA protein binds specifically at the consensus sequence DnaA box within guaB. This box may therefore function as a transcriptional terminator. A computer search of nucleotide sequences in the EMBL data library (release 30, 1992) for the E.coli chromosome revealed 48 sites conforming to the consensus sequence for the DnaA box. These sites are located non-randomly on the chromosome map with a bias towards the origin of replication (oriC). A model is proposed whereby genes containing DnaA boxes (including guaB) comprise a DnaA regulon. Transcriptional activity of these genes may be regulated by the cellular concentration of the DnaA protein.
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Published date: 1992
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Local EPrints ID: 461869
URI: http://eprints.soton.ac.uk/id/eprint/461869
PURE UUID: 202a05ac-43dc-437d-8de0-c31ca74b29f7
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Date deposited: 04 Jul 2022 18:57
Last modified: 04 Jul 2022 18:57
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Author:
Fisehaye Tesfa-Selase
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