Ishwar Radhakrishnan, PhD
Associate Professor of Biochemistry, Molecular Biology and Cell Biology
Weinberg College of Arts and Sciences
Research Interests:
Large molecular assemblies comprising multiple protein and/or nucleic acid subunits perform a variety of specialized tasks in the cell. Research in the Radhakrishnan laboratory is directed towards clarifying the structural and thermodynamic bases of protein-protein and protein-DNA interactions that regulate the formation and/or recruitment of these molecular assemblies. Our current efforts are focused on the structural and functional analysis of binary and ternary complexes using biochemical and biophysical (primarily, solution state NMR) approaches. These studies address issues relating to the sequence and structural requirements for complex formation, including defining the molecular determinants of affinity, cooperativity, and specificity. An important adjunct to these studies is the discovery of novel functions. Molecular Mechanisms of Recruitment, Assembly, and Action of Coregulator Complexes in Transcriptional Repression (sub-title in bold) A major ongoing research project in the lab involves a comprehensive characterization of interactions mediated by the Sin3 corepressor. Sin3 is a key scaffolding protein of a large (~2 MDa), evolutionarily conserved, multi-protein complex that is required for normal growth and development of tissues and organisms ranging from yeast to human. Sin3 physically associates with multiple components of the complex including chromatin-modifying activities such as histone deacetylases (HDACs), whose actions lead to alterations in the histone code and to concomitant changes in transcriptional status, and a surprisingly large number of seemingly unrelated and structurally diverse transcription factors. Members of the Sin3 complex ostensibly lack intrinsic DNA-binding activity, but are thought to be recruited to specific regions of the genome by sequence-specific DNA binding transcription factors and chromatin-binding proteins to effect gene silencing. Our early studies focused on the molecular basis for the apparent promiscuity of Sin3 interactions with transcription factors while more recent studies center on other components of the complex with particular emphasis on interactions with chromatin. Molecular Mechanisms of Ubiquitin Recognition (sub-title in bold) Another major project that has been ongoing for several years is the characterization of molecular interactions involving monomeric ubiquitin (monoubiquitin). Ubiquitin chains covalently linked to cellular proteins constitute a well-characterized signal for proteosome-mediated degradation. Over the past decade, additional non-traditional roles for conjugated ubiquitin in endosomal sorting, gene regulation, intra-nuclear localization, and budding of retroviral virions have emerged. The ubiquitin signal is transmitted through direct physical interactions with a variety of ubiquitin-binding motifs found in proteins that participate in the aforementioned processes. More than a dozen such motifs have been described thus far and over half of these have been implicated in efficient and accurate targeting of ubiquitinated membrane proteins to the destination organelle during endocytosis. Collaborative Mechanisms in Transcription Regulation (sub-title in bold) Many eukaryotic sequence-specific DNA binding transcription factors act collaboratively to regulate transcription of specific genes. We are elucidating the structural basis for the synergistic activation of an ovarian gene (inhibin-α) – an important hormone in female reproductive physiology – mediated by SF-1, a member of the nuclear hormone receptor (NHR) superfamily, CREB, a cyclic AMP responsive factor, and GATA-4, a member of the GATA family of transcription factors. SF-1 is an important regulator of several ovarian genes and in many cases the protein appears to functionally interact with transcription factors that confer cyclic AMP responsiveness to the respective promoters. Unlike many better-characterized NHRs, SF-1 binds to a wide range of DNA sequences as a monomer. SF-1, CREB, and GATA-4 are thought to cooperatively assemble on the inhibin-α promoter creating a higher-order protein-nucleic acid structure that serves as the platform for the recruitment of coactivator complexes. The long-term goals of this project are to define the structural basis for the molecular interactions that lead to the assembly of the higher-order structure and the subsequent recruitment of chromatin-modifying complexes.
Selected Publications:
Sahu, S.C., Swanson, K.A., Kang, R.S., Huang, K., Brubaker, K., Ratcliff, K., and Radhakrishnan, I. (2008). Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor. J. Mol. Biol. 375, 1444-56.
He, Y., Hicke, L., and Radhakrishnan, I. (2007). Structural basis for ubiquitin recognition by SH3 domains. J. Mol. Biol. 373, 190-6.
Little, T.H., Zhang, Y., Matulis, C.K., Weck, J., Zhang, Z., Ramachandran, A., Mayo, K.E., and Radhakrishnan, I. (2006). Sequence-specific DNA recognition by steroidogenic factor 1: A helix at the C-terminus of the DNA binding domain is necessary for complex stability. Mol. Endocrinol. 20, 831-43.
Swanson, K.A., Hicke, L., and Radhakrishnan, I. (2006). Structural basis for monoubiquitin recognition by the Ede1 UBA domain. J. Mol. Biol. 358, 713-24.
Swanson, K.A., Knoepfler, P.S., Huang, K., Kang, R.S., Cowley, S.M., Laherty, C.D., Eisenman, R.N., and Radhakrishnan, I. (2004). HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations. Nat. Struct. Mol. Biol. 11, 738-746.
