ENZYMATIC MODIFICATION OF THE 5' END OF RIBONUCLEIC ACID; STUDIES OF RNA CAPPING AND RNA PHOSPHORYLATION IN VITRO
SHUMAN, STEWART HOWARD
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Studies on the enzymatic modification of the 5' terminus of ribonucleic acid--with particular attention to those reactions involving RNA capping and methylation, RNA 5' phosphorylation and 5' (gamma) phosphate removal--are presented in this thesis as summarized below.;RNA guanylyltransferase, an enzyme which catalyzes transfer of GMP from GTP to the 5' triphosphate or diphosphate terminus of RNA to generate the cap structure G(5')ppp(5')N has been purified from vaccinia virions. This enzyme is but one component of a multifunctional capping enzyme complex containing RNA triphosphatase and RNA (guanine-7) methyltransferase activities. The capping enzyme complex has a sedimentation coefficient of 6.5, an apparent native Mr of 120,000 and consists of two polypeptides of Mr 95,800 and 26,400.;The present demonstration that RNA guanylyltransferase catalyzes a GTP:PPi exchange reaction suggested that the guanylylation reaction consists of a series of partial reactions involving a capping enzyme-GMP intermediate. This thesis substantiates the existence of a vaccinia capping enzyme-GMP complex and demonstrates its involvement as an intermediate in the capping reaction. The characteristics of this and other reactions mediated by the vaccinia enzyme complex have been studied and are reported herein.;5' Hydroxyl polyribonucleotide kinase, an enzyme which catalyzed the phosphorylation of 5' hydroxyl ends of RNA in the presence of ATP, has been isolated from extracts of HeLa cell nuclei. The kinase requires a divalent cation for activity, has an alkaline pH optimum and is sensitive to sulphydryl antagonists. 5' Hydroxyl terminated polydeoxyribonucleotides are phosphorylated much less efficiently than the 5' hydroxyl terminated polyribonucleotides, and the kinase preparation is inactive on ribonucleoside-3'-monophosphate. The sedimentation coefficient of the kinase is estimated to be 5.6S. The possible role of this activity in RNA processing in vivo is dicussed.