NMR studies of protein interactions with nucleic acids : translation termination and transcription.
Date of Issue2012
School of Biological Sciences
My thesis includes three subprojects related to NMR-based studies of three protein classes involved in DNA/RNA recognition. My focus is to use the advanced solution NMR methods to answer critical biological questions complementing collaborative efforts within each individual subproject. In the first subproject I studied structural changes in a stop-codon recognizing protein induced by mutations altering stop-codon recognition specificity. Ribosome is one of the most sophisticated supramolecular complexes in the cell. Some of the processes that occur within this machinery include translation initiation, elongation, and termination, all of which involve coding mRNA, a set of aminoacyl-tRNAs, and various protein factors acting in a concerted manner. Translation termination requires class I release factors: RF1 and RF2 in Bacteria, and eRF1 in Eukarya. eRF1 is able to decode all three stop codons. Until now, the mechanism of stop codon recognition by eRF1 leading to hydrolysis of the ester bond on the peptidyl tRNA remains obscure. We working as a team at Prof. Pervushin’s lab (Li Yan and Leo Wong) proposed a model of the binding mode of eRF1 to pre-translation termination ribosomal complex based on our solution structures of wild-type and several mutants of eRF1’s N-domain as well as its interactions with model RNA and M- and C- domains of the same protein. Due to the global character of structural perturbation induced by mutations, Residual Dipolar Coupling (RDC) data were obtained using 15N labeled sample aligned with Pf1 phages. The RDC measurements obtained were then applied in the structure calculation and refinement of the eRF1 mutant Q122FM(Y)F126 using structure calculation software. The most interesting mutant, Q122FM(Y)F126 restricts the decoding capability of eRF1 to UGA codon only. From the 3D structures, we established that the mutations alter conformation and dynamics of the GTS loop distant from the sites of mutations. Based on published biochemical and mutagenesis studies, we propose that the GTS loop forms a switch that allows reading of the multiple codons. NMR analysis revealed that helix α1 of N-domain interacts specifically with double-stranded RNA with a bulge or internal loop resembling a mismatch of H44 in 18S rRNA of Eukaryotic ribosome. From these results, a 3D model of eRF1 interactions with ribosome in pre-termination state is proposed. In my second project we collaborated with Dr. Ralf Jauch in order to provide structural basis for activity of novel drug candidates interfering with transcription factor/cognate DNA interactions. The DNA binding domains of transcription factors have so far been considered too impervious to be tackled as drug targets although upregulated transcription factors are a major cause of cancer and other diseases. Here we identified a Dawson-POM as an unconventional but potent compound to inhibit the DNA binding activity of Sox2. We used NMR to locate binding site of the drug candidates on Sox2. The mode of interaction of the Dawson-POM with the Sox2-HMG domain involves predominantly electrostatic interactions at the pocket just outside of the DNA binding region, but still adequately positioned to compete with the negatively charged DNA backbone. In summary, the inhibitory mechanism demonstrated here could eventually spawn the development of modified classes of POM based drugs to specifically combat aberrant gene expression. The objective of the third project is to assess the structural flexibility of N-terminal tail domain of Histone 4 when it is in the compact nucleosome array using deuterium/protons exchange experiment in NCP. 15N labeled Histone 4 was prepared and 2D [1H-15N]-TROSY series of experiments were carried out for protein recovered from reconstituted NCPs in H2O followed by controlled exposure to D2O. The sequential assignment of the backbone H4 was used to identify the dynamics of the NCP as well as arrays in the compact form. We observed that that although in compact states, the magnitude of protection is not as pronounced as expected. This suggests that the NCP is still flexible, albeit being in a compact state. The first two projects are either prepared or published. The last project is in active development.