The evolution and composition of RNA polymerase IV in plants

Thesis (Ph. D.)--University of Washington, 2006.This thesis explores two aspects of the evolution of RNA polymerase in plants. In both cases, components of the RNA polymerase II transcription system of the nucleus were found to have undergone duplicative proliferation.In the derived angiosperm taxa Ericales and Lamiidae, two forms exist for the gene RPB2, encoding the second-largest subunit of RNA polymerase II. Other angiosperms possess only a single form. Chapter 2 of this thesis shows that the two paralogous forms of RPB2 evolved as separate lineages following a duplication event early in eudicot evolution. In the vast majority of taxa, one or the other form of RPB2 has been lost from the genome. This pattern of duplication and loss, which the data show applies to other nuclear genes as well, may well be responsible for the rapid diversification of flowering plants that occurred during the Paleocene and Eocene.At a much earlier time, prior to the existence of land plants, the ancestral plant genes for RNA polymerase II protein subunits underwent duplication of a very different sort to encode the enzyme we know as RNA Polymerase IV. This thesis presents comparative sequencing studies and phylogenetic analyses of the genes for the largest Pol IV subunit (RPD1 and RPE1) and for the second-largest Pol IV subunit (RPD2). The results clearly show that both of these Pol IV subunit genes were derived from the corresponding Pol II homologs. These duplication events were separated in evolutionary time, with RPD1 appearing in charalian algae and RPD2 first appearing in primitive land plants.Extensive purification was carried out with nuclear RNA Polymerase preparations from cauliflower, toward the goal of identifying a catalytically active RNA Polymerase IV. Owing to the low level of this protein, no activity could be demonstrated. Nonetheless, by using immunochemical techniques and mass spectrometric analysis, it was shown that RPD1/RPE1 and RPD2 proteins are closely associated and that the RPB3 gene product, together with common subunits are part of the Pol IV apoenzyme. Pol IV is non-essential for normal growth and development of Arabidopsis. However, Pol IV knockout mutants are defective in asymmetric DNA methylation