Research Project
7061644
DESCRIPTION (provided by applicant): Leishmania is a parasitic protozoan (order Kinetoplastida) that causes a spectrum of disease ranging from asymptomatic to lethal, resulting in widespread human suffering and death. LmjF presents atypical mechanisms of gene expression, since transcription seems to initiate in only a few regions per chromosome, generating long polycistronic transcripts that are processed by trans-splicing to produce mature mRNAs. Little is known in kinetoplastids about transcription by RNA Polymerase III (Pol III), which transcribes several conserved and abundant small RNAs (such as tRNAs, 5S rRNAs and snRNAs) that play critical roles in cell metabolism. Our main objective is to characterize Pol III promoters and transcriptional complexes in Leishmania major Friedlin (LmjF), whose genome sequence has been recently completed. The specific hypothesis is that Pol III promoters and transcription factors in LmjF (and other kinetoplastids) differ considerably from those present on other eukaryotes. The specific aims are to: 1. Analyze transcription of Pol III genes in LmjF. Transcription start sites will be mapped by 5'-RACE, and termination sites localized by RT-PCR with poly(A)-tailed RNA. Nuclear run-on analysis will be performed with single-stranded DNA fragments spanning coding and intergenic regions. Promoter activity will be tested by transient-transfection studies. 2. Examine the DNA-protein interactions between the Pol III promoters and the transcription machinery. DNA-protein interactions will be analyzed by electrophoretic mobility shift assays. DNase I footprinting assays will be carried out to identify the specific DNA sequences that interact with the Pol III complex. 3. Identify the protein components of the Pol III complex in LmjF. The tandem affinity purification (TAP-tag) method will be used to purify Pol III transcriptional complexes. The protein components in these complexes will be identified by mass-spectrometry analysis.
