Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a ‘Slacked-Dynamic’ state throughout the observation window where many of them have switched between multiple ‘Wobbling States’ in the course of attainment of packaged form. A range of DNA ‘Flexure Angles’ has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes. This study opens up an opportunity to improve the understanding of the functions of other nucleoid-associated proteins (NAPs) by complementing the previous detailed atomic-level structural analysis, which eventually will allow accessibility towards a better hypothesis.
Single-molecule
Förster resonance energy transfer microspectroscopic
methods are employed for real-time monitoring and to gain deeper insights
into the formation of the polypurine reverse Hoogsteen hairpin (PPRH)
and its triplex-forming activity. The heterogeneity in the behavior
of individual PPRHs has been documented, and it is seen that the degree
of anharmonic plasticity of the antiparallel hairpin is stabilized
by the formation of reverse Hoogsteen (RH) bonds. While being involved
in the hairpin formation, they flip reversibly between the open and
closed conformations, irrespective of the concentration of ions present
in their microenvironment. However, the nature of the cation present
in the buffer plays a crucial role in determining the structural stability.
The Watson Crick (WC) bonds are found to be more dynamic in the triplex
compared to that of the RH base pairs, indicating the involvement
of progressive WC bonds during the triplex motif formation by the
PPRH. The majority of the intact triplex DNA attained a semifolded
relaxed state before progressing toward a tightly folded state, emphasizing
the fact that the folding mechanism pursues an ambiguous path in the
mode of acquiring the final step of the triple helix motif. Moreover,
the presence of triplex-forming sequences in the regulatory regions
of the genome further provides an intricate link between the experimental
results and sequence occurrence.
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