Department of Chemistry, University of California, Berkeley 94720.
J Mol Biol 205: 291-314 (1989)
Abstract
During transcription, Escherichia coli RNA polymerase is capable of
removing the nucleotide that it has just added to a growing RNA chain,
and this removal depends on the presence of small concentrations of
pyrophosphate. Chemically, the removal reaction is simply the reversal
of the incorporation reaction, and we have observed the generation of
free triphosphate as a result. After the removal the enzyme can continue
synthesis. To test whether this reaction can provide an error
correction mechanism, misincorporation rates were measured at a single
position in an RNA transcript by withholding the correct nucleotide for
that position, measuring the amount of readthrough transcript, and
analyzing the readthrough transcripts with nearest-neighbor analysis and
enzymatic RNA sequencing. The removal of pyrophosphate increases the
rate of misincorporation. We present a theory that explains how
reversible incorporation can increase the available discrimination free
energy between correct and incorrect nucleotides and therefore may
increase the fidelity of transcription. The formation of a covalent
phosphodiester bond allows discrimination on the basis of helical
structure as well as base-pairing. We propose that the important
discrimination step is the translocation of the enzyme from one site on
the DNA template to the next, and that reversible incorporation is
necessary in order to take full advantage of the maximum discrimination
free energy.
Mesh Headings
Unique Identifier: 89178649
Chemical Identifiers (Names)