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Free keywords:
gene transcription; NTP binding; RNA polymerase;
transcription initiation
Abstract:
During transcription initiation by RNA polymerase (Pol) II,
a transient open promoter complex (OC) is converted to
an initially transcribing complex (ITC) containing short
RNAs, and to a stable elongation complex (EC). We report
structures of a Pol II–DNA complex mimicking part of the
OC, and of complexes representing minimal ITCs with 2, 4,
5, 6, and 7 nucleotide (nt) RNAs, with and without a nonhydrolyzable
nucleoside triphosphate (NTP) in the insertion
site þ1. The partial OC structure reveals that Pol II
positions the melted template strand opposite the active
site. The ITC-mimicking structures show that two invariant
lysine residues anchor the 30-proximal phosphate
of short RNAs. Short DNA–RNA hybrids adopt a tilted
conformation that excludes the þ1 template nt from the
active site. NTP binding induces complete DNA translocation
and the standard hybrid conformation. Conserved
NTP contacts indicate a universal mechanism of NTP
selection. The essential residue Q1078 in the closed trigger
loop binds the NTP 20-OH group, explaining how the
trigger loop couples catalysis to NTP selection, suppressing
dNTP binding and DNA synthesis.
