טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentGross Lital
SubjectPossible Roles of Post-translational Modifications of
Rpb4/7 in Regulating Gene Expression
DepartmentDepartment of Medicine
Supervisor Professor Mordechai Choder
Full Thesis textFull thesis text - English Version


Abstract

The yeast Rpb4/7 plays roles in all the major stages of the mRNA life cycle: regulation of transcription, mRNA export, translation and mRNA decay.

Rpb4/7 binds RNA polymerase II (Pol II) thus stimulating transcription, and binds the transcripts co-transcriptionally and then escorts the mRNA throughout its life. Previously, we found that it modulates each step that the mRNA undergoes by virtue of its capacity to interact with key regulators temporally and spatially. We proposed that it is capable of integrating all stages into a system (the "mRNA coordination" concept). Using 2 dimensional gel electrophoresis (2DE), we showed that Rpb4/7 exists in various isoforms due to post-translational modifications (PTMs). About 20 distinct spots were identified by 2DE of Rpb4 and 3 spots by 2DE of Rpb7. This provoked us to examine whether these PTMs are involved in its capacity to integrate all stages into a system.  As the first stage toward this goal I examined whether the PTMs are stage specific. Indeed, we found that the PTMs change as Rpb4/7 progresses from one stage to the other.  Specifically, in the context of transcribing Pol II, Rpb4/7 seems to be carry only two modifications; in the context of the translation complex it carries 3 different modifications, while in the context of the decay machinery it is carry one modification. Some of the modifications are phosphorylation. Most modifications are dependent on prior binding to Pol II. These modifications seem to be biologically significant because they are responsive to environmental conditions, such as heat shock. Using mass spec, we tentatively identified lysine (K) and glutamic acid (E) methylations, K- gly-gly modifications (an indication for ubiquitination or neddylation) and K acetylation in Rpb7p. K- gly-gly modifications, E, K and aspartic acid (D) methylations and K acetylation were identified in Rpb4p. Until now, these modifications were not tested by methods other than mass spec.

We propose that PTMs play roles in quality controlling the various stages of gene expression and in the capacity of Rpb4/7 to integrate all stages of gene expression into a system.