During gene transcription, RNA is synthesized from a DNA template by DNA-dependent RNA polymerases (Pols). Eukaryotic cells contain three nuclear RNA polymerases, responsible for the synthesis of ribosomal RNA (Pol I), pre-messenger RNA (Pol II) and small RNAs including transfer RNAs (Pol III). The three RNA polymerases differ in subunits composition.
Whereas Pol II has 12 subunits, Pol III consists of 17 subunits ranging from 10 to 160 kDa. With a total molecular weight of nearly 700 kDa, Pol III is the largest and the most complex nuclear RNA polymerase. Of the 17 Pol III subunits, ten are unique to Pol III, five are common to all three eukaryotic RNA polymerases, and two are shared by Pol I and III.
Whereas the crystal structure of Pol II is known, and a n electron microscopic structure of Pol I is available, there is no structural data on Pol III, despite its important roles in regulating cell growth and differentiation.
Here we propose to study the three-dimensional structure of the Pol III enzyme by X-r ay crystallography, to obtain novel insights into the mechanisms of transcription, the specific features of Pol III structure and function compared to the Pol II system, and into the evolution of the transcription machineries.
Pol III will be purified from yeast strains, and crystals of the entire complex will be sought with the use of a nanoliter robot. Complexes of Pol III with initiation factors and nucleic acids will be prepared for future analysis by cryo-electron microscopy. The studies will benefit from the expertise on Pol II structure determination in the institute.
A structural description of Pol III will be of central importance to the large community concerned with gene expression and its regulation, and will contribute to the further development of structural biology techniques. The longer-term goal of this project is to unravel the determinants for promoter specificity of the three nuclear polymerases.
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