Mechanistic studies on transcription activation via DNA looping in a prokaryotic promoter-enhancer system

In this thesis, transcription activation was studied in a prokaryotic promoter-enhancer system. It comprises the E. coli RNA polymerase, which is associated with the alternative sigma factor sigma54 (RNAP-sigma54), and the transcription activator protein NtrC (nitrogen regulatory protein C), which binds to a remote enhancer region to the promoter. Enhancer-bound NtrC contacts the RNA polymerase at the promoter by means of DNA looping (closed complex) and induces DNA melting of the promoter DNA by RNAP-sigma54 (open complex). The following aspects of this process were studied: (1) Three different sigma54-specific promoter sequences were analyzed in binding studies and in in vitro transcription experiments. These promoters were known to have different overall promoter strength as determined by in vivo expression and DNA footprinting studies. Since initiation of transcription comprises different subsequent steps (promoter-binding by RNAP-sigma54, isomerization to the open complex and formation of a stable elongation complex) it was still unclear, which is the rate limiting step of the total reaction. For the glnAp2 and nifL promoters, the promoter-binding by RNAP-sigma54 was rate limiting. In contrast, for the nifH promoter with a high affinity to RNAP-sigma54 but with a low in vivo expression level, the DNA melting step determined the overall speed of the transcription initiation reaction. (2) By scanning force microscopy it was determined that promoter-bound RNAP-sigma54 bends the DNA and is for this reason preferably localized in the end-loop of a supercoiled DNA, since the DNA is more bent in this region. The localization in the end-loop facilitates the interaction between NtrC and RNAP-sigma54 in spite of the low flexibility of the intervening DNA. (3) The interaction between sigma54 and NtrC was studied in an ATPase assay and in gel shift experiments. It was shown that sigma54 has no effect on the ATPase activity of NtrC under the experimental conditions whereas enhancer-binding of NtrC strongly stimulates the ATPase activity by facilitating the oligomerization of NtrC. Binding studies that were performed by analytical ultracentrifugation and gel shift experiments have also shown that sigma54 alone only weakly bind sthe promoter DNA in contrast to the RNAP-sigma54 holoenzyme. This supports the idea, that the sigma factor acts by recognizing the promoter sequence whereas the RNAP holoenzyme provides the binding energy for high affinity promoter binding. (4) In vitro transcription experiments showed that NtrC activates with different efficiency and can even act as a repressor depending on the position, number and arrangement of its binding sites. Certain combinations of weak and strong NtrC binding sites were shown to activate transcription from the promoter at very different concentrations of NtrC. This enables a regulation of transcription in dependence of the NtrC concentration. From these results, a model of RNAP-sigma54-NtrC-mediated transcription activation was developed: Accordingly, the proximal NtrC sites very close to the promoter facilitate the interaction between activator and RNA polymerase in a loop complex at low NtrC concentrations, whereas at higher concentrations the transcriptional activation is limited to a maximum level. In this case, a NtrC species is formed, which can interact with the RNAP-sigma54 without DNA looping.