Search

Regulation of protein mobility is a fundamental aspect of cellular processes. In this study, we examined the impact of DNA methylation on the diffusion of nucleoid associated protein Hfq. This protein is one of the most abundant proteins that shapes the bacterial chromosome and is involved in several aspects of nucleic acid metabolism. Fluorescence microscopy was employed to monitor the movement of Hfq along double-stranded DNA, which was stretched due to confinement within a nanofluidic channel. The mobility of Hfq is significantly influenced by DNA methylation. Our results underscore the importance of bacterial epigenetic modifications in governing the movement of nucleoid associated proteins such as Hfq. Increased levels of methylation result in enhanced binding affinity, which in turn slows down the diffusion of Hfq on DNA. The reported control of protein mobility by DNA methylation has potential implications for the mechanisms involved in target DNA search processes and dynamic modelling of the bacterial chromosome.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher β-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

The effect of the heat unstable nucleoid structuring protein HU on the conformation of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. Pre-incubated DNA molecules contract in the longitudinal direction of the channel with increasing concentration of HU. This contraction is mainly due to HU-mediated bridging of distal DNA segments and is controlled by channel diameter as well as ionic composition and strength of the buffer. For over-threshold concentrations of HU, the DNA molecules compact into an condensed form. Divalent magnesium ions facilitate, but are not required for bridging nor condensation. The conformational response following exposure to HU was investigated with a nanofluidic device that allows an in situ change in environmental solution conditions. The stretch of the nucleoprotein complex first increases, reaches an apex in ∼20 min, and subsequently decreases to an equilibrium value pertaining to pre-incubated DNA molecules after ∼2 h. This observation is rationalised in terms of a time-dependent bending rigidity by structural rearrangement of bound HU protein followed by compaction through bridging interaction. Results are discussed in regard to previous results obtained for nucleoid associated proteins H-NS and Hfq, with important implications for protein binding related gene regulation.

Nucleoid associated proteins (NAPs) play a key role in the compaction and expression of the prokaryotic genome. Here we report the organisation of a major NAP, the protein H-NS on a double stranded DNA fragment. For this purpose we have carried out a small angle neutron scattering study in conjunction with contrast variation to obtain the contributions to the scattering (structure factors) from DNA and H-NS. The H-NS structure factor agrees with a heterogeneous, two-state binding model with sections of the DNA duplex surrounded by protein and other sections having protein bound to the major groove. In the presence of magnesium chloride, we observed a structural rearrangement through a decrease in cross-sectional diameter of the nucleoprotein complex and an increase in fraction of major groove bound H-NS. The two observed binding modes and their modulation by magnesium ions provide a structural basis for H-NS-mediated genome organisation and expression regulation.

Recent discoveries of noncoding regulatory RNAs have led to further understanding of the elements controlling genetic expression. In E. coli, most of those ncRNAs for which functional knowledge is available were shown to be dependent on the Hfq RNA chaperone and to act as inhibitors of translation by base pairing with their mRNA target. Nevertheless, there are also some examples where the sRNA plays a role of a translational activator, structurally enhancing ribosome binding to mRNA. In this work, we seek to understand the dynamics of DsrA-based positive regulation of rpoS mRNA, encoding the σ(S) RNA polymerase subunit, and to understand how it helps to mitigate environmental stress in bacteria. Our analysis is based on the first absolute quantification of the copy number of both the sRNA and of its corresponding mRNA in combination with mathematical models for post-transcriptional regulation. We show that on average, DsrA is present at a ratio of 3 to 24 copies per cell, while an rpoS transcript is present at a level of 1 to 4 copies per cell, both levels increasing when temperature is decreased. Our analysis supports the idea that temperature dependency of DsrA degradation is not a crucial condition for the attainment of observed DsrA steady levels, but highlights that this may have a marked influence on the dynamics of the regulation, notably to speed up the time of recovery to normal RNA levels after ending the stress signal. Further, our analysis also reveals how reversibility of RNA complex formation and σ(S)-regulated degradation act to reduce intrinsic noise in σ(S) induction. Taking into account the importance of this master regulator, which allows E. coli as well as other important pathogens to survive their environment, the present work contributes to complete the panel of multiple signals used to regulate bacterial transcription.

Hfq is a bacterial protein involved in RNA metabolism. Besides this, Hfq's role in DNA restructuring has also been suggested. Since this mechanism remains unclear, we examined the DNA conformation upon Hfq binding by combining vibrational spectroscopy and neutron scattering. Our analysis reveals that Hfq, which preferentially interacts with deoxyadenosine rich sequences, induces partial opening of dA-dT sequences accompanied by sugar repuckering of the dA strand and hence results in a heteronomous A/B duplex. Sugar repuckering is probably correlated with a global dehydration of the complex. By taking into account Hfq's preferential binding to A-tracts, which are commonly found in promoters, potential biological implications of Hfq binding to DNA are discussed.