Objective
RNA polymerase is the principal enzyme of gene expression and atarget for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. The broad objective of this project is to understand the molecular mechanism of RNA polymerase function and regulation in relation to its structure using a combination of biochemical and moleculargenetic approaches.
To this end, insertions, deletions and genetic splits will be generated in the cloned rpoB and rpoC genes of E.coli which encode the two largest subunits of RNA polymerase. The mutant subunits and their fragments will then be over expressed from rpo expression plasmids, and the mutant enzymewill be isolated from cells or reconstituted from individual subunits and their fragments in vitro. The malfunctioning of RNA polymerase caused by the mutations will be characterized using anarsenal of in vitro assays which allow to monitor and discriminate between individual steps of RNA polymerasefunctional cycle.
Particular emphasis will be given to the analysis of the interactions of the mutant enzymes with promoter DNA, functional defects of the mutant enzymes during the inchworming cycle of transcription elongation, and regulatory defects in factor-dependent transcription. This approach is anticipated to provide molecular details of promoter binding, the catalytic reaction and the mechanism of elongation, and will help identify target sites for regulatory factors.
The experiments with deletions and subunit fragments are expected to dissect the two large subunits of RNA polymerase into individual domains. Protein-protein interactions of these domains during RNA polymerase assembly process as well as in transcription complexes will be studied using a combination of formaldehyde crosslinking, neutron scattering, fluorescence energy transfer measurements and surface plasmon resonance.
The results of this work are expected to provide a better view of the general architecture of RNA polymerase and transcription complexes and will become an important resource for direct structural studies and molecular-modeling. The expected results of the proposed researchactivities will be published in the peer-reviewed international scientific journals.
Topic(s)
Call for proposal
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82152 Martinsried bei München
Germany