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Single Molecules of RNAP II: Backtracking and Beyond

Dr. Eric Galburt
MPI Molecular Cell Biology and Genetics, Dresden


RNA polymerase II (RNAP II) is responsible for transcribing all mRNAs
in eukaryotic cells in a highly regulated process that is conserved from
yeast to human and that serves as a central control point for cellular
function.  We have been investigating the transcription dynamics of
RNAP II molecules in the presence of opposing force in the presence and
absence of TFIIS, a transcription elongation factor known to increase
transcription through nucleosomes.  Using a single-molecule dual-trap
optical-tweezers assay we found that the response of RNAP II to force is
entirely determined by enzyme backtracking, a process where the enzyme
slides upstream on the DNA template, thus losing the ability to convert
chemical energy into mechanical work and becoming a simple brownian
particle.  RNAP II molecules ceased to transcribe and were unable to
recover from backtracks at a force of 7.5 ± 2 pN, only one third of the
force determined for E. coli RNAP. We show that distributions of
pause lifetimes imply that backtracked RNAP II molecules diffuse in
discrete base-pair steps and suggest that backtracks may account for
most of RNAP II pauses.  Interestingly, addition of TFIIS allowed
transcription to proceed up to much higher forces.  Taken together,
these results describe a potential regulatory mechanism of transcription
elongation in eukaryotes by which transcription factors modify the
mechanical performance of RNAP II, allowing it to bypass transcriptional
barriers present in vivo.