NMR application and development : study of the ribosomal RNA-L11 complex
Author(s)Ilin, Serge, 1977-
Nuclear magnetic resonance application and development : study of the ribosomal RNA-L11 complex
Massachusetts Institute of Technology. Dept. of Chemistry.
Harald J. Schwalbe.
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The L11 protein of the large ribosomal subunit binds to a highly conserved domain of the 23S rRNA and jointly with RNA mediates GTPase activity of elongation and release factors. It consists of a C-terminal domain, which is the main anchor and determinant for the binding to rRNA and an N-terminal domain. The N-terminal domain plays only a limited role in RNA binding and is thought to be involved in interactions with elongation and release factors as well as with the antibiotics thiostrepton and micrococcin. As part of the doctoral dissertation the NMR solution structure of the full-length L11 protein from the thermophilic eubacterium Thermotoga maritima in its free form was solved, the binary complex of L 11 protein with RNA and the N-terminal domain alignment were investigated, the possible inhibition mode of the L11-RNA complex by thiostrepton was proposed and studies aimed at NMR method development and application were undertaken. The structure of L11 is based on a large number of orientational restraints derived from residual dipolar couplings in addition to conventional NOE-based restraints. The solution structure of L11 demonstrates that in contrast to many other multi-domain RNA- binding proteins, the relative orientation of the two domains is well defined as evidenced both by heteronuclear ¹⁵N relaxation data and residual dipolar coupling data. Comparison to the X-ray structure of L11 bound to its cognate RNA-domain reveals that RNA-binding not only induces a rigidification of a flexible loop in the C-terminal domain but a sizeable reorientation of the N-terminal domain. The domain orientation in the free L11 shows some similarity to that of ribosome bound L11 in complexes with EF-G. Upon binding to RNA based on chemical shift mapping, the most affected regions are localized in the disordered loops between helix [alpha]3 and [beta]-sheet [beta]4 and between helices [alpha]5 and [alpha]6, which are both located on the C-terminal domain. The N-terminal domain on the other hand shows no significant contacts with RNA in contrast to the proposed strong contacts observed in the crystal structure of the complex. The orientation of the N-terminal domain based on the residual dipolar coupling alignment shows a similarity to the L11 structure solved in the large ribosomal subunit of Deinococcus radioduransa. The localization and conformation of the thiostrepton antibiotic in the ternary complex with RNA and L11 protein resolved by NMR restraints such as chemical shift mapping and residual dipolar couplings is consistent with the biochemical data such as footprinting and point mutations. Thiostrepton seems to lock the complex in the EF-G*GDP state. Therefore the GTP- hydrolysis is allowed to occur but the release of EF-G, GDP and inorganic phosphate are inhibited, confirming previously reported kinetic data. Based on the chemical shift data, thiostrepton orients itself with the protruding polypeptide chain into the inside of the complex, where it can adjust its conformation to fill the space between RNA and L11. Such a complex formation explains how thiostrepton can form a binary complex with the RNA but not with L 1, however in the ternary complex, L11 greatly increases the binding of the thiostrepton by locking it into place. As a part of the doctoral dissertation, several other projects were also undertaken: the study of non-natural pyranosyl-RNA structures and its analogues and the development of a novel NMR method to determine the conformation around the O-glycosidic linkage based on ³J-(C, H) couplings and C-H-dipolar cross-correlated relaxation.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Includes bibliographical references (p. 173-189).
DepartmentMassachusetts Institute of Technology. Dept. of Chemistry.
Massachusetts Institute of Technology