A proteomic approach to the study of Neurospora crassa ribosomal dynamics
EMSL Project ID
5402
Abstract
We propose to apply the LC/MS/MS global proteomics approach to study eukaryotic ribosome dynamics using the model filamentous fungal organism, Neurospora crassa. The experimental tractability and recently sequenced and annotated genome make N. crassa an ideal candidate in which to apply proteomic methods to interesting and relevant biological problems. Translation of mRNA into protein by ribosomes is a fundamental biological process and the experiments proposed here have the potential to open new avenues of research in this area by mapping factors associated with ribosome at different stages of translation. It is possible peptides identified in these studies that were previously annotated as ?hypothetical protein? will be associated with a function. Our initial experiment will focus on global peptide identification from ribosomes biochemically purified from N. crassa cultures. We hope to identify a core set of ribosomal proteins and strongly associated factors in this experiment. We will use second set of experiments to identify additional factors important for translation, i.e. those that associate with the ribosome on a dynamic or variable basis. We can his-tag near the N-termini of peptides and purify them as nascent peptides associated with the ribosome and we can also purify ribosomes based on association with biotinylated mRNA. In these experiments, various classes of genes will be his-tagged (potential classes include genes encoding secreted proteins and primary and secondary metabolite production enzymes). Following a nutritionally or chemically induced stalling of translation, stalled ribosomes that were translating his-tagged proteins will be purified on an affinity column and subjected to global proteomic peptide identification. Depending on the stage of translation and the class of his-tagged protein, we plan to identify a wide range of peptides that associate with ribosomes under various conditions.
Project Details
Project type
Exploratory Research
Start Date
2003-12-01
End Date
2005-04-11
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members