Gene expression in Trypanosoma brucei

Aim: To provide quantitative data that will allow modeling of gene expression for all enzymes of redox metabolism and the pentose phosphate pathway. Modeling will be used to predict enzyme levels based on the integration of an RNA degradation model with translation and protein degradation rates.

Plan: The amounts of a protein in a cell can be determined by the rates of transcription, mRNA processing, translation, mRNA turnover and protein degradation. In trypanosomes analysis is simpler because the transcription rates of individual genes are (with few exceptions) not subject to control (Clayton and Shapira, 2007). This investigation will involve two main studies:

1. Determination of the abundance, processing efficiency, and degradation kinetics of the mRNAs encoding all enzymes of interest. For this goal RNA will be extracted at several times points of trypanosomes treated with Actinomycin D and Sinefungin, to inhibit RNAprocessing and transcription, and sequenced with Next Generation Sequencing (RNAseq).

2. Determination of the rate of translation. This will be based on ribosomal loading: we will fractionate extracts on sucrose gradients and perform RNASeq across the polysome profile. From this we can predict protein synthesis rates.

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Created: 24th Feb 2012 at 13:19

Last updated: 20th Nov 2014 at 09:34

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