Alfonso Mondragón DNA topoisomerases, catalytic RNA molecules, molecular basis of spectrin flexibility

Research Interests  

DNA topoisomerases. The long term goal of our work is to understand the catalytic mechanism of these molecules in atomic detail. In particular, we are interested in understanding how these enzymes perform complex topological rearrangements of DNA molecules. DNA topoisomerases are of interest for several reasons: 1) they are responsible for maintaining the topological state of DNA and are involved in a variety of crucial cellular processes. 2) Their involvement in key processes has lead to the development of drugs whose target is topoisomerases. 3) Topoisomerases catalyze a complex reaction that involves cutting and resealing the DNA and passing DNA strands through this break. These reactions are not easy to visualize or understand. The structures of several topoisomerases and fragments of them have truly led to a near-atomic picture of the way a very complex reaction is catalyzed. 4) Topoisomerases are excellent examples of complex molecular machines that perform a complicated reaction in the cell. Type I enzymes work in the absence of an external energy source, such as ATP, and for this reason present an opportunity to understand a process where the energy to drive large domain movements is harnessed from the energy stored in the DNA, and 5) The structural studies may provide the information to develop new chemotherapeutic agents.

In the last few years our laboratory has worked on the structure of several different type I topoisomerases, including E. coli DNA topoisomerases I and III (type IA), and vaccinia virus and Deinococcus radiodurans topoisomerase I (type IB), and more recently onMethanopyrus kandleri topoisomerase V. In all cases, a combination of structural and biochemical work has helped elucidate the atomic basis of the catalytic mechanism of these enzymes.

Catalytic RNA molecules. A second large research area is the structure of large RNA molecules and, in particular, RNase P.   RNA plays a pivotal role in biology as it is involved in many cellular processes. It is also unique amongst nucleic acids in being able to perform chemical catalysis. In the cell, RNA molecules can self-cleave or process other RNA molecules in a manner that up to a few years ago was completely unknown and unexpected. These findings have led to the idea of a pre-biotic RNA-world, where RNA molecules were the first molecules to appear. The discovery of the catalytic properties of RNA molecules has also rekindled the interest in these molecules both from a basic and from an applied point of view.

RNase P is one of only two ribozymes conserved in all three kingdoms of life and is required in the 5' maturation of all tRNAs. In the last years, we solved the crystal structures of the specificity domain of Bacillus subtilis and Thermus thermophilus RNase P and also of the intact RNA component of T. maritima RNase P. In the structure of the intact molecule, the entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition, and the location of the active site. A model with bound tRNA is in excellent agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme. This is the first structure of an A-type bacterial RNase P solved and represents one of the largest RNA molecules whose structure is known.

In the future we plan to continue our work on RNase P. Our immediate goals are to obtain structures of the holoenzyme and a tertiary complex involving the RNA component, the protein component, and pre-tRNA.

Spectrin. Proteins of the spectrin superfamily are designed for the vital task of providing cells with a deformable skeleton and a flexible matrix. Members of this ubiquitous family, such as a-spectrin and dystrophin, are long molecules formed by tandem repeating units of 106-109 amino acids, each folded into a triple-helical coiled-coil.   Understanding of the relative arrangement of the repeats, the nature of the linker region between them, and the general disposition of the repeats is crucial to further our knowledge of spectrin flexibility. To address these questions, we solved the structure of several related molecules formed by two or three repeats of a-spectrin.   The structures show that spectrin has an ordered a-helical linker region, that the relative arrangements of the repeats can vary, and that the repeats can rearrange themselves. The structures allowed us to propose two possible models for spectrin flexibility, the first models based on atomic data.

We are now also focusing our attention on repeats of human spectrin that contain the region involved in interactions with other cytoskeletal proteins such as ankyrin. We are employing a combination of biophysical and structural approaches with the long-term goal of understanding the atomic basis of the interaction of spectrin and other cellular proteins.

Selected Publications

An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerasesGunn KH, Marko JF, and Mondragón A. Nature Structural & Molecular Biology. 2017 May;24(5):484-490.

Allosteric transcriptional regulation via changes in the overall topology of the core promoterPhilips SJ, Canalizo-Hernandez M, Yildirim I, Schatz GC, Mondragón A, and O'Halloran TV. Science. 2015 August 21;349(6250):877-881.

Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex. Clark MD, Marcum R, Graveline R, Chan CW, Xie T, Chen Z, Ding Y, Zhang Y, Mondragón A, David G, and Radhakrishnan I.  PNAS. 2015 July 14;112(28):E3669-E3678.

Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III.  Terekhova K, Marko JF, and Mondragón A. Nucleic Acids Research. 2014 October 13;42(18):11657-11667.

Structure and function of the T-loop structural motif in noncoding RNAs.  Chan CW, Chetnani B, and Mondragón A. Wiley Interdisciplinary Reviews: RNA. 2013 September/October;4(5):507-522.

View all publications by Alfonso Mondragón listed in the National Library of Medicine (PubMed). Current and former IBiS students in blue.