Title

RNA polymerase I in Trypanosoma brucei

Date of Completion

January 2012

Keywords

Biology, Genetics|Biology, Parasitology|Health Sciences, Human Development

Degree

Ph.D.

Abstract

Trypanosoma brucei is a tsetse-transmitted, lethal, protistan parasite of humans harboring a unique multifunctional RNA polymerase (pol) I that transcribes ribosomal genes and units encoding its major cell surface antigens VSG and procyclin. In this versatile RNA pol I system the enzyme is recruited to different promoter types and sequestered into two subnuclear compartments. Hence, we hypothesized that the trypanosome RNA pol I system essentially depends on parasite-specific proteins/protein domains (chapter 1). Previous analyses showed that trypanosomes possess 10 of 12 core RNA pol I subunit homologs; only orthologs of the yeast RPA43/RPA14 doublet remained elusive. Instead, published evidence suggested that the RNA pol II paralog of RPA43, RPB7, assumed the essential RPA43 function in trypanosomes. However, work described in chapter 2 unambiguously demonstrated that these claims are false and that RNA pol I transcription does not require RPB7. ^ The multifunctional aspects of RNA pol I are particularly relevant in the mammal-infective bloodstream form (BF). However, a key obstacle for analyzing this process in BFs has been the lack of an in vitro transcription system. Here we developed a small-scale procedure that yielded transcriptionally active BF extract accommodating the low BF density obtainable in culture. This system was optimized, validated, and employed to demonstrate life cycle-independent functionality of the class I transcription factor A (CITFA; chapter 2). ^ Since it was now possible to investigate the transcriptional role of unique proteins in BFs, a characterization of the BF RNA pol I complex was undertaken. Rat infections were used to grow genetically modified BFs to large numbers and RNA pol I was tandem affinity-purified via the tagged subunit RPB6z. We identified 30 new proteins that co-purified with RPB6z and potentially have BF-specific roles in transcription. We also found that RNA pol I had been partially deactivated possibly by glucose deprivation of BFs during the lengthy harvesting procedure (chapter 4). ^ Finally, the technological progress in this project enabled me to collaborate with Nguyen et al. in the characterization of CITFA (appendix 1) and with Kim et al. to characterize the trypanosome MCM-BP complex involved in VSG silencing (appendix 2). ^