The expression of genes is tightly controlled, predominantly at the point of transcription. RNA polymerase (RNAP) must first bind to a deoxyribonucleic acid (DNA) promoter upstream of a gene to transcribe it. However, the ability of RNAP binding is dictated by the core promoter DNA sequence, the presence of transcription activator or repressor proteins and numerous other factors. The strength of promoters has been indirectly measured. Only a few studies have attempted to directly address the RNAP flux through transcription units, and further studies are still required. In the current study, the aim was to directly correlate RNAP gene transcription with the strength of core promoter elements. To do that, I employed the direct method of chromatin immunoprecipitation (ChIP), followed by quantification of immunoprecipitated DNA. For promoter regions, this method directly measures the occupancy by RNAP; for regions within transcription units, the flux of the RNAP was deduced. A range of semisynthetic promoters, with different combinations of core promoter elements to obtain different levels of expression, was used to validate our method. This direct method enabled the calculation of “promoter competitivity”, “promoter occupancy index” (POI), RNAP “escape index” (EI), “fragment occupancy percentage” (FOP) and the time interval between transcribing RNAPs (Tint). On the basis of Tint, the number of RNAPs crossing any DNA sequence of interest per second (polymerase per second; PoPS) was calculated. Surprisingly, the results of the present study revealed that the RNAPs are well separated during transcription of the \(lac\) operon.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:715704 |
| Date | January 2017 |
| Creators | Sendy, Bandar |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/7576/ |
Page generated in 0.0015 seconds