Expanding Crystallization Tools for Nucleic Acid Complexes Using U1A Protein Variants

Publication date: Available online 15 February 2020Source: Journal of Structural BiologyAuthor(s): Hannah Rosenbach, Julian Victor, Jan Borggräfe, Ralf Biehl, Gerhard Steger, Manuel Etzkorn, Ingrid SpanAbstractThe major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR- and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes.Graphical abstract

Source: Journal of Structural Biology - February 17, 2020 Category: Biology Source Type: research