Abstract
Collisions between paused transcription elongation complexes and replication forks inevitably happen, which may lead to collapse of replication fork and could be detrimental to cells. Bacterial transcription factor DksA and its cofactor alarmone ppGpp were proposed to contribute to prevention of such collisions, although the mechanism of this activity remains elusive. Here we show that DksA/ppGpp do not destabilise transcription elongation complexes or inhibit their backtracking, as was proposed earlier. Instead, we show, both in vitro and in vivo, that DksA/ppGpp increase fidelity of transcription elongation by slowing down misincorporation events. As misincorporation events cause temporary pauses, contribution to fidelity suggests the mechanism by which DksA/ppGpp contribute to prevention of collisions of transcription elongation complexes with replication forks. DksA is only the second known accessory factor, after transcription factor Gre, that increases fidelity of RNA synthesis in bacteria.
Highlights
Multi-subunit DNA-dependent RNA polymerase (RNAP) is highly processive and can continue RNA synthesis for thousands of nucleotides without dissociation from the template DNA or the RNA product
Oligonucleotides were from Integrated DNA Technologies (IDT). ppGpp was from TriLink BioTechnologies
We tested the effects of DksA and ppGpp on transcription elongation on a well studies template containing T7A1 promoter [8,36,40]
Summary
Multi-subunit DNA-dependent RNA polymerase (RNAP) is highly processive and can continue RNA synthesis for thousands of nucleotides without dissociation from the template DNA or the RNA product. Pausing can be caused by signals in the nucleic acids of the elongation complex (EC) that impede incorporation of nucleoside monophosphate (NMP) by altering properties of the active centre (elemental pauses) [2,3] or slow down forward translocation by RNAP (translocation pauses) [4,5]. Either of these pauses can lead to backtracking of RNAP, a phenomenon when the 3 end of RNA disengages from the active centre and RNAP shifts backward along the template. While elemental and translocation pauses are usually relatively short-living, backtracking, if not resolved, frequently represents a dead-end event [8]
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