Dr. Remus T. Dame

Vrije Universiteit
Division of Physics & Astronomy / FEW
De Boelelaan 1081
1081 HV Amsterdam

Tel. + 31-(0)20-59 87838
Room T 0.60
E-mail: rtdame@nat.vu.nl
Homepage


Prokaryotic genome organization and transcription regulation

The structural effects of H-NS and HU, as important both for the global organization of DNA and for the role of these proteins in the modulation of transcriptional activity, have been previously investigated using scanning force microscopy and biochemical techniques. These studies have provided significant information as with regard to the properties of these proteins. H-NS is a protein, which has the ability to form bridges between adjacent DNA helices. On one hand this is at the basis of the compaction of DNA by H-NS (1), on the other hand it has important implications for the role of this protein in the regulation of transcription. H-NS recognizes curved DNA, which is often found close to promoter sequences of H-NS repressed genes, and this seems to involve bridging of DNA around the apex of the curve (2). Repression of transcription by H-NS then is a consequence of the RNA polymerase molecule being occluded from the promoter sequence. In addition, these binding properties are at the basis of a particular mechanism of repression of transcription at the rrnB P1 promoter. At this promoter RNA polymerase can still bind to the promoter in the presence of H-NS and, in fact, the binding of both proteins simultaneously occurs in a cooperative fashion. Repression then is a consequence of RNA polymerase being held trapped within a repression loop, which is a result of the DNA aside of the RNA polymerase being bridged (3). Studies on the structural effects of HU suggest that this protein is not involved in the compaction of DNA, but rather that it functions as antagonist of H-NS mediated compaction (4).

These observations illustrate the versatility of the H-NS protein and support the notion that a lot of different mechanisms may explain resgulation at different promoters. The elucidation of these mechanisms requires further biochemical and microscopical characterizarion. In addition, a lot of dynamic aspects of the regulatory mechanism of these and other nucleoid-associated proteins (such as IHF and Fis) are still unknown. The aim of our studies is to gain insight into such aspects of the various mechanisms of transcription regulation employed by this type of proteins. To that purpose the role of these proteins in the regulation of transcription is also addressed using single molecules techniques such as optical tweezers.

References:

Dame RT, Wyman C and Goosen N.
H-NS mediated compaction of DNA visualised by atomic force microscopy.
Nucleic Acids Res. 2000 Sep 15;28(18):3504-10.

Dame RT, Wyman C and Goosen N.
Structural basis for preferential binding of H-NS to curved DNA.
Biochimie. 2001 Feb;83(2):231-4.

Dame RT, Wyman C, Wurm R, Wagner R and Goosen N.
Structural basis for H-NS-mediated trapping of RNA polymerase in the open initiation complex at the rrnB P1.
J Biol Chem. 2002 Jan 18;277(3):2146-50.

Dame RT and Goosen N.
HU: promoting or counteracting DNA compaction?
FEBS Lett. 2002 Oct 9;529(2-3):151

R.T. Dame, G.J.L. Wuite
On the role of H-NS in the organization of bacterial chromatin. From bulk to single molecules and back
Biophys. J. 85 (2003) 4146-4148

R.T. Dame, C. Wyman, N. Goosen
Insights into the regulation of transcription by scanning force microscopy
J. of Microscopy (2003) 244-253

J. van Noort, S. Verbrugge, N. Goosen, C. Dekker, R.T. Dame
Dual architectural roles of HU: formation of flexible hinges and rigid filaments
Proc. Natl. Acad. Sci. USA 101 (2004) 6969-6974