All biological processes take place in highly crowded cellular environments. However, the effect that molecular crowding agents have on the folding and catalytic properties of RNA molecules remains largely unknown. Here, we have combined single-molecule fluorescence resonance energy transfer (smFRET) and bulk cleavage assays to determine the effect of a molecular crowding agents on the folding and catalysis of a model RNA enzyme, the hairpin ribozyme. Our single-molecule data reveal that PEG favors the formation of the docked (active) structure by increasing the docking rate constant with increasing PEG concentrations. Furthermore, Mg2+ ion-induced folding in the presence of PEG occurs at concentrations ∼7-fold lower than in the absence of PEG, near the physiological range (∼1 mM). Lastly, bulk cleavage assays in the presence of the crowding agent show that the ribozyme’s activity increases while the heterogeneity decreases. Our data is consistent with the idea that molecular crowding plays an important role in the stabilization of ribozyme active conformations in vivo.

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.

Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family of remodellers. We demonstrate that the binding energy for CHD4-nucleosome complex formation -even in the absence of nucleotidetriggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4-6 base-pair steps. This decoupling of entry-and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. We propose a mechanism for nucleosome sliding based on these and published data.