Use of the broccoli aptamer to study transcription in vitro and in vivo

G.E. Folkers, Willem Kegel

Project description

In a shared project with Willem Kegel we are working toward methodology to be able to quantify the role of the total accessible number of DNA base pairs in transcription regulation in biological cells . With ‘accessible DNA base pairs’ we mean the part of the total genome that is accessible for transcription factors and DNA polymerase. While the direct influence of transcription factors on transcription regulation is now well understood, transcription regulation might further involve the accessible amount of non-regulatory DNA. That amount varies during the cell cycle, is mediated by various DNA structuring proteins, and couples to environmental conditions. Transcription factors and RNA polymerase mainly reside onto non-specific DNA and variations in the total accessible amount of DNA influence transcription rates in a non-trivial manner.

We have established a protocol to directly monitor transcription activity in vitro using an RNA aptamer that upon binding of a dye becomes fluorescent. With this method we can address the above mentioned questions by adjusting promoter sequences with respect to the strength of the promoter, the inhibitor and combine this with the concentration of competing (non-specific) DNA

Research proposal

The first task will be to obtain a set of reporter constructs with promoters of various strength and one or more lac repressor binding sites adjacent to the promoter sequence. The activity of these constructs in the presence or absence of various concentrations of inhibitors will be analysed in vitro. Subsequently the amount of non-specific DNA will be analysed using non-specific DNA as competitor. Finally the affinities of both the repressor and the polymerase will be determined in vitro using thermophoresis and fluorescence anisotropy. By comparing the accumulation of the mRNA using the broccoli we intend to use in collaboration with Willem Kegel a biophysical description of gene regulation. With this joined effort we hope to understand which additional factors contribute to tight regulation of gene expression observed in vivo

Methods used:

  • Recombinant DNA technology including PCR, cloning, site directed mutagenesis, sequencing
  • Protein expression and protein purification
  • Biophysical characterization of binding affinity using e.g. fluorescence anisotropy, thermophoresis