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Abstract:
We show that RNA polymerase (Pol) II prevents erroneous
transcription in vitro with different strategies
that depend on the type of DNA,RNA base mismatch.
Certain mismatches are efficiently formed but impair
RNA extension. Other mismatches allow for RNA
extension but are inefficiently formed and efficiently
proofread by RNA cleavage. X-ray analysis reveals
that a T,U mismatch impairs RNA extension by forming
a wobble base pair at the Pol II active center that
dissociates the catalytic metal ion and misaligns the
RNA30 end. The mismatch can also stabilize a paused
state of Pol II with a frayed RNA 30 nucleotide. The
frayed nucleotide binds in the Pol II pore either
parallel or perpendicular to the DNA-RNA hybrid
axis (fraying sites I and II, respectively) and overlaps
the nucleoside triphosphate (NTP) site, explaining
how it halts transcription during proofreading, before
backtracking and RNA cleavage.
