University of Turku
Supervisor: Georgiy Belogurov
In all life forms, the genetic information coded in DNA sequence is transcribed by RNA polymerases (RNAP). Multi-subunit RNAPs are versatile enzymes that, beside their polymerizing activity, are able to unwind DNA double helix, motor along DNA as a thermal ratchet, shift to reverse motion, and hydrolyze nascent RNA. As a consequence of their central role in gene expression, multi-subunit RNAPs are conserved among Bacteria, Eukaryotes and Archaea. Eukaryotes possess three paralogous RNAPs that are specialized to transcribe distinct subsets of genes. Pol I and Pol III read RNA coding genes wheras protein coding genes are read by Pol II. We investigate the functional specialization of RNAPs and try to understand how different needs in transcriptional read-out are realized by differences in RNAP functions.
Transcription, the synthesis of RNA from DNA template, is a multi-step process where RNAPs are subjected to regulation by many protein factors, sequence elements and small molecules. The second line of research focuses to the features of transcription elongation cycle and its regulation. During elongation, RNAP selects templated NTP substrates and incorporates them to the 3´ end of nascent RNA. Upon binding of cognate NTP, a mobile element called "trigger loop" folds and closes the active site for catalysis. Catalysis is followed by trigger loop unfolding and the release of reaction by-product pyrophosphate. Before addition of next nucleotide, RNAP needs to translocate along DNA by one base. Elongation cycle is frequently interrupted by regulatory and stochastic pauses that influence gene expression. We study the mechanics and thermodynamics of different steps in the elongation cycle by using both biochemical and biophysical approaches to dissect various RNAP activities. The details of the molecular mechanisms in transcription are elucidated in in vitro systems using newly developed fluorescent methods along with more traditional transcription assays.