Application Notes

The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA

Issue link:

Contents of this Issue


Page 0 of 1

1 Characterization of Protein - Nucleic Acid Interactions Application Note NT-MO-016 The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA Dr. Thomas Schubert Lab of Prof. Gernot Längst, University of Regensburg in cooperation with 2bind GmbH, Germany Abstract Histone proteins are associated with DNA and form a nucleoprotein structure termed chromatin. The chromatin structure allows the eukaryotic genome to be stored and transported but also needs to be accessible to various crucial DNA dependent processes Thus, histone modifying, DNA modifying and chromatin binding proteins specifically interact and regulate chromatin.. Interestingly, many of the chromatin modifying proteins also display RNA binding capacity. Here we characterize the specific interaction of the Decondensation factor 31 (Df31) with nucleic acids. Df31 was recently shown to form be part of a RNA dependent regulating mechanism influencing chromatin structure and function. Introduction In eukaryotic cells the DNA is packed into chromatin by the association of histone molecules and non-histone proteins. Various non-histone proteins influence the structure of chromatin and thus also the function of chromatin. A class of proteins called chromatin remodelling complexes are capable to move nucleosomes along the DNA, thereby revealing or hiding transcription factor binding sites. Other protein classes change the functionality of chromatin by the modification of chromatin with epigenetic marks like methylation or acetylation. Interestingly, many of these proteins involved in chromatin remodelling and modification have the capacity to bind RNA directly or interact with complexes that contain RNA (Mattick et al., 2009). The binding of HP1, a major structural component of heterochromatin, for example, is highly dependent on the presence of RNA (Maison et al., 2002). Moreover, DNMTs and DNA methyl binding proteins recognize and interact with RNA (Jeffery and Nakielny, 2004). Interestingly, only a few of these chromatin remodelling proteins are described to possess a preference for particular DNA sequences. However the modifications have to be purposefully directed to "different positions, in different loci, in different cells" (Mattick et al., 2009), implying another layer of information to guide these processes, which seem to be RNA based. In a recent study the Drosophila Decondensation factor 31 (Df31) was shown to be involved in an RNA dependent mechanism regulating chromatin compaction (Schubert et al., 2012). To determine whether Df31 is capable to bind nucleic acids and especially RNA MicroScale Thermophoresis analyses were performed. Results In this application note, the binding behavior of Df31 to nucleic acids was evaluated, using MicroScale Thermophoresis. Fam or Cy5-labeled random ssRNA, ssDNA, dsDNA molecules of same sequence and length (29 nt) were used in a concentration of 50 nM in the experiments. A serial dilution of Df31 was added with starting concentration of 680 µM to the corresponding nucleic acid. The samples were filled into standard capillaries and incubated at 27 °C for 15 min, prior to the MST measurement. For the assay three independent measurements were performed. A clear binding curve for the interaction of the Df31 protein and ssRNA could be detected. However, ssDNA and dsDNA showed no binding to Df31.

Articles in this issue

view archives of Application Notes - The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA