Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongation

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Abstract
Current models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre- and post-translocated states. Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor. Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e. low K(m)). The addition of an extra nucleotide destabilizes the repressor-operator roadblock leading to an increase in transcriptional readthrough. Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e. high K(m)s) by specifically increasing their intracellular concentrations. Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding. They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level.