We are studying variations in the mechanical conformations induced by the nucleoid-associated protein HU. Two different approaches are being used. The first one relays on ‘minimal intervention’ which is obtained by using a method called Tethered Particle Motion (TPM) which is a method that doesn’t require the use of external forces, thus decreasing the interruption to the biological process. Basically, a surface-tethered molecule (linked to a nanobead at the other end) diffuses in a restricted volume in a physiological solution and the bead’s scattered light is detected with optical microscopy. The second approach is to find the structural details of the HU-complexes and for that we use Atomic Force Microscope (AFM).
The DNA contains all the genetic information. In Eukarayotes the DNA is found inside the nucleus. The size of a human cell is ~10-100 μm while the size of a fully stretched DNA is about 1.5m. This difference raises important questions such as how could it be accessible for transcription and for a wide range of proteins that act on the DNA? How do proteins find their specific target sequence in the DNA?
We are exploring some of these questions which are both important scientifically, and can influence our understanding for health-related issues. In one of the projects we track the variations in the size of the DNA persistence length induced by the nucleoid-associated protein HU (Histone-like from strain U93).
The HU protein, which is one of the most abundant proteins in prokaryotes, was known until recently to compact the DNA. However, recent FRET studies (Stavans, J. Mol. Biol. 341, 419–428, 2004) and single molecule experiments using magnetic and optical tweezers (Skoko, Biochemistry 43, 13867-13874, 2004; Van Noort, PNAS, 101, 6969–6974 ,2004) claim that when increasing the protein concentration to a few hundreds nano molar, the protein stiffens the DNA, thus increasing its persistence length.
We use two different approaches to detect variations in the dynamics and conformations of single DNA molecules due to HU binding. Tethered Particle Motion (TPM) allows us to detect the dynamic variations in the end-to-end distance of single DNA molecules due to the bending/unbending activity of HU. In our TPM setup a small nanobead is attached to a linear dsDNA at one end while the other end is attached to the surface. Illuminating the sample makes it possible to detect the scattered light from the gold nanobead, a higher signal to noise ratio is achieved thanks to using darkfield illumination. The bead diffuses in a restricted volume which varies in the presence of a protein.
The second approach is to find the structural details of the HU-DNA complexes using Atomic Force Microscope (AFM). We find the angles distribution for different concentrations and calculate the CDF.
We found that at relatively low HU concentration (up to 500 nM) the DNA is significantly more flexible than without the protein. Increasing the concentration up to few micromolars leads to a more rigid form of the DNA, however not as rigid as without any protein. Taking into account our CDF plot obtained from the AFM data, we adopt the model of Rappaport and Rabin (PRL 2008) which states that binding of proteins to two neighboring segments leads to local stiffening of the DNA whereas binding to only one segment leads to bending of the DNA. We therefore conclude that HU induces local bending/stiffening on the DNA which leads to incoherent persistence length along the DNA.
- M. Lindner, G. Nir, H. R. C. Dietrich, I. T. Young, E. Tauber, I. Bronshtein, L. Altman and Y. Garini, Studies of single molecules at their natural form, Israel Journal of Chemistry 49, 283-291 (2009).
- G. Nir, M. Lindner, H. R. C. Dietrich, O. Girshevitz, C. E. Vorgias, and Y. Garini, HU protein induces incoherent DNA persistence length, Biophysical Journal 100, 784-790 (2011).