Date of Award
Doctor of Philosophy (PhD)
Dr. Thomas Neilson
The sequence and conformation of short RNA strands was investigated by proton nuclear magnetic resonance spectroscopy. This physical technique gives conformational information about RNA strands in solution at the nucleoside level. The short oligoribonucleotides used in these studies were chemically synthesized by the phosphotriester method developed by T. Neilson and associates, which is capable of preparing a wide variety of sequences in the quantities required for NMR spectroscopic experiments and the many in vitro termination assays. The work presented in this thesis was the first to demonstrate that NMR spectral information can be used to sequence RNA oligomers and that sequence and conformation around termination codons can redeflne their activity in vitro.
A set of parameters has been developed that provides a new and accurate mathematical approach for the assignment of the NMR chemicaI shifts for heterobase ring and anomeric protons in oligoribonucleotides. The set of empirical shift parameters was derive from the chemical shifts of RNA oligomers and can also be applied to DNA species. The accuracy of the parameters is such that they can be used to accurately determine the sequence of RNA oligomers.
The specificity of in-vitro prokaryotic chain termination was examined as a function of the sequence neighbouring UAA. RF-1 dependent termination was assayed from the release of N-acetyl or N-formyl-methionine from initiation complexes containing N~acetyl- or N-formyl-met-tRNA:ribosomes: AUG or AUG (N)m. In the AUG case, a second oligomer was added to cause release.
When added to the AUG-bound intermediate, hexamer UAAUAG, containing tandem termination codons, was slightly more effective in the release of N-acetyl-met-tRNA by RF-1 than UAA, UAAN (where N is A, G, C, or U), and UAAUGA whereas UAAUAA, was least effective. Oligomers, AUGUA, AUGUAA, AUGUUA, AUGUUAA, AUGUUAU and AUG(U)mUAA(A)₁₈_₂₅ (where m = 1-5) stimulated binding of N-acetyl-met-tRNA and release of N-acetyl-met by RF-1, whereas AUGCUA, AUGCUAA and AUGUAA(A)n, were less effective.
The sequence UA acts as release signal for RF-1, its activity varying with mRNA sequence contrast. The conformation surrounding UA is responsible which is dependent upon the nature of adjacent nucleoside residues. This UA recognition is competed for by cognate aminoacyl-tRNAs.
Some of the results presented in this thesis have appeared in the literature:
1. Alkema, D., Bell, R., Hader, P. A., and Neilson, T. (1981) in Biomolecular Stereodynamics" Ed., Sarma, R. H., Adenine Press, NY.
2. Alkema, D., Bell, R., Hader, P. A., and Neilson, T (1981) J. Am Chem. Soc. 103, 2866-2868.
3. Alkema, D., Hader, P. A., Bell, R. A., and Neilson, T. (1981) Biochemistry 21, 2109-2117.
4. Hader, P. A., Neilson, T., Alkema, D., Kofoid, E.C., and Ganora, M. C. (1981) FEBS Letters 136, 65-69.
5. Hader, P. A., Alkema, D., Bell, R.A., and Neilson, T. (1982) J. Chem. Soc., Chem Commun. 1017.
Hader, Paul Anthony, "¹H-NMR Studies of Sequence and Conformation Using Short RNA Oligomers" (1982). Open Access Dissertations and Theses. Paper 1331.