Cryo-electron microscopic studies of RNA polymerase complexes

Related RNA polymerases (RNAPs) carry out gene transcription all
three domains of life. This thesis deals with the structure
determination of RNAPs and their functional complexes from
different species. Protein complexes were preserved in their native
state in aqueous solution, imaged by cryo- transmission electron
microscopy and structural models were obtained using the single
particle reconstruction method. New and physiologically relevant
insights into RNAPs subunit architecture, the general transcription
mechanism and its regulation were gained. The structure of an
archaeal RNA polymerase identified similarities to its eukaryotic
counterpart, RNA polymerase II. The conservation of the overall
enzyme architecture as well as the close resemblance of structural
elements and functional surfaces needed for basic transcription
mechanisms underlines the evolutionary relationship between
archaeal and eukaryotic RNAPs. The comprehensive study of RNA
polymerase III and its regulation by Maf1 gave profound insights
into the molecular basis of how eukaryotic transcription is
shutdown under stress conditions to ensure cell survival. Maf1
binds RNAP III at its clamp domain and rearranges a specific
subcomplex needed for interaction with the initiation factor Brf1.
This specifically impairs binding of RNAP III to its promoters and
inhibits transcription initiation. Furthermore, it was demonstrated
that Maf1 binds to RNAP III that is already engaged in
transcription elongation, thus leaving activity intact but
preventing re-initiation. Taken altogether, these results converge
on the essential mechanism of RNAP III-specific transcription
repression by Maf1.