Fluorescent Modifications of Transfer Ribonucleic Acids

178 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.Modifications of transfer ribonucleic acids (tRNA's) have been accomplished by chemical and enzymatic methods. The chemical modification has involved the reaction of methylmalondialdehyde (MMDA) with E. coli tRNA('Phe) with the hope of extending the use of substituted malondialdehydes and investigating their applicability as probes of the structures and functions of RNA molecules. 1,N('2)-(2-Methylallylidene)guanosine ((mu)-guanosine) is the product of MMDA and guanosine. This compound has been characterized by ultraviolet, fluorescence, and nuclear magnetic resonance spectroscopy. We have shown that reaction of MMDA with E. coli tRNA('Phe) occurs and that, at least to some extent, products with fluorescent properties similar to those of (mu)-guanosine are formed. The primary site of modification has been rigorously identified as either the X-nucleoside or the 7-methylguanosine in the variable loop of tRNA('Phe). The modified tRNA('Phe) can be aminoacylated to the extent of 20% as compared with the unmodified tRNA('Phe).The enzymatic modification has involved the introduction of fluorescence into yeast tRNA('Phe) by replacing the 3'-terminal adenosine residue with a fluorescent analog of this nucleoside, linear-benzoadenosine. This has been accomplished through the addition of linear-benzoadenosine 3',5'-bisphosphate to the 3'-end of shortened yeast tRNA('Phe) via a T4 RNA ligase-catalyzed reaction, followed by alkaline phosphatase removal of the resultant 3'-phosphate group. A method has been devised that will unambiguously define the sequence of nucleotides at the 3'-end of tRNA's modified in this region of the molecule. This procedure involves ribonuclease U(,2) digestion of the tRNA, followed by separation of the digestion products by electrophoresis on DEAE paper. The labelled oligonucleotides are identified by comparing their electrophoretic mobilities with those of marker oligonucleotides.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD