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Abstract:
Crystallographic studies of the RNA polymerase II
(Pol II) elongation complex (EC) revealed the
locations of downstream DNA and the DNA-RNA
hybrid, but not the course of the nontemplate
DNA strand in the transcription bubble and the
upstream DNA duplex. Here we used singlemolecule
Fluorescence Resonance Energy
Transfer (smFRET) experiments to locate nontemplate
and upstream DNA with our recently developed
Nano Positioning System (NPS). In the
resulting complete model of the Pol II EC, separation
of the nontemplate from the template strand at
position +2 involves interaction with fork loop 2. The
nontemplate strand passes loop b10-b11 on the Pol
II lobe, and then turns to the other side of the cleft
above the rudder. The upstream DNA duplex exits at
an approximately right angle from the incoming
downstream DNA, and emanates from the cleft
between the protrusion and clamp. Comparison
with published data suggests that the architecture
of the complete EC is conserved from bacteria to
eukaryotes and that upstream DNA is relocated
during the initiation–elongation transition.
