Abstract
RNA synthesis in all organisms is regulated throughout transcript initiation and elongation. The secondary channel (SC) of multi-subunit RNA polymerases (RNAPs) allows access to the enzyme active site and is a nexus for regulation. In E. coli, multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but how these factors function without interference and the dynamics of their interactions with RNAP are unclear. GreB is an SC protein that promotes endonucleolytic transcript cleavage in elongation complexes that are backtracked by nucleotide misincorporation. Here we used multi-wavelength single-molecule fluorescence microscopy to characterize in vitro GreB effects on transcript elongation and the dynamics of GreB interactions with elongation complexes. Contrary to previous dogma but consistent with structural data, we found that high GreB concentrations substantially slow transcript elongation. During steady-state elongation, GreB binds RNAP at near-diffusion-limited rates, but remains bound for <1s, approaching the duration of the nucleotide addition cycle and much shorter than needed to produce a complete mRNA. To examine whether GreB specifically selects backtracked complexes, we reconstituted static complexes stabilized in backtracked and non-backtracked configurations. By verifying the functional state of each molecular complex measured, we showed that both configurations bind GreB at rates similar to actively elongating complexes, and that backtracking does not kinetically stabilize bound GreB. Our results exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, they suggest that GreB binds rapidly and randomly to all elongation complexes to patrol for those requiring nucleolytic rescue, and that its short residence time serves to minimize inhibition of non-backtracked elongation complexes and to prevent interference with the function of other SC factors.