Melissa S. Jurica
Structural Approaches To Large Macromolecular Complexes
My lab uses the tools of structural biology to understand how large macromolecular complexes carry out their biochemical functions. An example of such a complex is the spliceosome, the multi-megadalton machine that removes the non-coding intervening sequences (introns) littering the pre-messenger RNA (pre-mRNA) transcripts of most eukaryotic genes. The spliceosome is a complicated entity, consisting of 5 RNAs and on the order of 100 different proteins in a dynamic assembly. The complex must precisely recognize and excise long intronic sequences in nascent transcripts and then ligate the coding sequences (exons) to generate mRNAs that correctly encode for proteins. Structurally, relatively little is known about the spliceosome or its components that provides insight into the assembly and catalytic mechanism of this complex, although inroads to structural understanding are now being generated.
The large size and complexity of many macromolecules provides several challenges to structure determination. With the spliceosome, the task is even more formidable due to its dynamic assembly, lack of symmetry and possible structural flexibility. It is therefore necessary in the case of such complicated machines to combine various structural techniques to create architectural representations. In our lab, we primarily use the tools of cryo-electron microscopy (cryo-EM) and X-ray crystallography to address these issues. Cryo-EM, where complexes preserved at liquid nitrogen temperature are directly imaged, is suited to large macromolecules available in limited quantities. Two-dimensional images of the molecules are computationally processed to generate a 3D reconstruction. We use this technique in combination with gold-labeling of components to provide ultrastructural information for splicing complexes. X-ray crystallography is used to determine high-resolution structures of individual spliceosome components. Our goal is to exploit the strategies of structural genomics to analyze the large number of spliceosomal proteins. Results from these endeavors can be combined with computational modeling and experimentally derived biochemical restraints to build highly detailed 3D models of the spliceosome that will be useful in furthering our understanding of the mechanistic underpinnings of pre-mRNA splicing. This multi-faceted approach can also be applied to other macromolecular complexes.