Position-dependent modulation of promoter activity in Escherichia coli

Bryant, Jack Alfred (2013). Position-dependent modulation of promoter activity in Escherichia coli. University of Birmingham. Ph.D.

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Abstract

Bacterial gene expression is primarily regulated at the initial step of transcription initiation.
This regulation has traditionally been viewed as due to the DNA sequence of promoters and factors that bind to them. However, the bacterial chromosome is a highly compacted and ordered structure. To date, the effects of position within the chromosome on promoter activity have barely been investigated.
Here, I report that activity of a single promoter, inserted at different loci in the
Escherichia coli chromosome, is dependent upon position within the chromosome.
Quantification of genomic DNA at a number of loci showed this was unrelated to gene dosage. Drastic changes in position-dependent hierarchies of promoter activity were observed on entry into stationary phase. However, no correlation between the orientation of a promoter, with respect to replication, and its activity was found.
Several factors are involved in this variation, including local sequence context. Increased promoter activity at origin-proximal loci was shown to be due to DNA supercoiling, maintained by the action of DNA gyrase. Transcription was shown to have a negative influence on the activity of downstream promoters. Further to this, the position of a promoter, with respect to a repressor-encoding gene, had an effect on its repression, further suggesting a role for genome organisation in the regulation of transcription. Finally, extended protein occupancy domains were shown to repress activity of inserted promoters, indicating that these domains act to silence the genes they encode. The combined data show that the position of a promoter within the E. coli chromosome plays a significant part in transcriptional regulation, and that structure of the chromosome is also likely to play a role.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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