During the past year, we have been focusing to develop new methodologies that are needed for studying the structural biology of the 3' UTR RNA of the human VEGF mRNA. These methods include GASR and G2G. GASR stands for """"""""derive global architecture from SAXS and RDC"""""""" data, and G2G stands for """"""""derive global structure of large RNAs in solution from global restraints"""""""". In addition, we have determined the three dimensional structure of the 3' UTR RNA fragment (102 nt) of the turnip crinkle virus (TCV). 1. Developing GASR method. Determining global structures of multi-subunit biomacromolecules in solution is a challenging problem. We report here a methodology that determines the global architecture of hetero- or homodimeric systems in solution using the residual dipolar couplings (RDCs) as the global orientation and the small-angle X-ray scattering (SAXS) as the global shape restraints; this methodology is implemented in an efficient program, known as global architecture derived from SAXS and RDC (GASR). We first applied the method to derive the global architecture of HIV protease, a globular homodimeric protein, to test the robustness of the method using simulated data with added noise. We then applied the method to determine the interfaces in the integrin-linked kinase (ILK)-PINCH complex using the experimental data. Without the benefit of SAXS data, the global architecture (and thus the interfaces of this complex) was underdetermined because of a lack of a sufficient number of detectable experimental distance restraints between the subunits of the complex. This method provides a new general approach for determining global structures of macromolecular complexes in solution. We have published the first manuscript on the method for an RNA:RNA complex (JACS, 2008 Mar 19;130(11):3292-3) and have submitted the second manuscript on this method. 2. Developing G2G method. We have developed a novel top-down methodology that uses duplexes global orientations and overall molecular dimension restraints, which were extracted from experimental NMR and small angle X-ray scattering (SAXS) data respectively, to determine global architectures of large RNA molecules. The methodology is implemented in an efficient computational program package, G2G. We demonstrate the efficiency of the method by rapidly determining the global structure of a 71-nucleotide RNA using experimental data. The backbone RMSD of the ensemble of the calculated global structures relative to the X-ray crystal structure is 3.0 0.4 , and this RMSD is only 2.4 0.16 for the three duplexes that were orientation- and translation-restrained during the calculation. The global structure dramatically simplifies interpretation of multi-dimensional nuclear Overhauser spectra for a rapid high resolution structure determination. We anticipate that this top-down method will become the standard routine for determining structures of large RNAs in solution. A manuscript about this method has been submitted. 3. Structure determination of the TCV RNA using NMR and small angle X-ray scattering (SAXS). The 3′ UTRs of many plant viral mRNAs have been demonstrated to have tRNA-like activities and have been proposed to fold into a tRNA-like structure. Among these plant viral RNAs, the 3′ UTR of the turnip yellow mosaic virus (TYMV) was best characterized biochemically, and a tRNA-like computational model based on tRNA has been proposed. However, a three-dimensional structure has not been determined for any of these UTRs. The experimentally determined tRNA-like structures will provide a critical piece of evidence to support the notion that the 3′ UTRs of these plant viruses are directly involved in the translation process and may be considered a conceptual step toward understanding the general mechanisms of 3′ UTR involvement in the regulation of gene expression. The tRNA-like structure of a 3′ UTR would also provide a critical piece of evidence supporting the notion that the function of these 3′ UTRs is related to their ability to form tRNA-like structures. The involvement of the 3′ UTR in translation may not be limited to plant viruses. Two other examples found in mammalian cells are the 3′ UTR of mRNAs coding for the ornithine decarboxylase (ODC) and the vascular endothelial growth factor (VEGF). In mammalian cells, the polyamines putrescine, spermidine, and spermine are essential for proliferation and differentiation. The mammalian ODC catalyzes the first and rate-limiting steps in the polyamine biosynthetic pathway. VEGF plays an important role in tumor angiogenesis and cancer progression. Both of these essential proteins are regulated at multiple levels, including the mRNA level, via 3′ UTRs. It has been proposed by Dr. Bruce Shapiros laboratory, of the CCR, that both RNAs share a secondary structure similar to that of the turnip crinkle virus 3′ UTR fragment (tcvRNA), and therefore, possibly the tertiary structure. Details of the structural determination of the tcvRNA are presented in a manuscript; the procedure is briefly described here. In the first step, we verified the secondary structure using the imino NOE-walk aided by the HNN-COSY hydrogen bond experiment and by the in-line probing experiments performed by Professor Anne Simons laboratory. We have also made numerous constructs to facilitate the imino assignments and to verify the proposed secondary structure. Because the tertiary interaction information is available for tcvRNA, the strategy for the structure determination is different from that used for the riboA RNA. The initial models were generated in two ways: (1) We assumed that all stems have standard A-form duplex structures, and the GAAA- tetraloop structure is the same as that reported . We generated the coordinates using the database built in my lab. The structural elements were then folded using tertiary base pairing predicted by Dr. Shapiro and verified by Simons laboratory. (2) Dr. Shapiros group has developed a computational program (RNA2D3D) for deriving a structural model based on the secondary and limited tertiary structural information, and has provided an initial structure of tcvRNA. We have used both the structural models as initial structures for the rigid-body refinement under the restraining of SAXS. The local structures of the duplexes and the tetraloop were kept rigid during the refinement. The initial refinement yielded an ensemble of structures that gave the best fit to the SAXS data. The Rg of the models is about 30.5 0.5 , compared with 31.1 of the experimental value. We are in the process of refining the structures with imino RDCs. The structure of tcvRNA is the first experimentally proven to fold into a tRNA-like structure. The tcvRNA binds to the P-site of the ribosome as a translation initiation enhancer. tcvRNA binds to the 80S ribosome specifically in the presence of phe-tRNA and acetylated phe-tRNA (AcPhe-tRNA), suggesting that tcvRNA competes with AcPhe-tRNA for the P-site. In conclusion, the results from our study provided the first example of a structured 3′ UTR fragment of an mRNA regulating translation initiation by binding to the ribosome. A similar role for a 5′ UTR mRNA has been demonstrated. We are compiling a manuscript of describing the structure determination of the tcvRNA.
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