Quantitation of DNA Binding Affinity Using Tethered Particle Motion.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered particle motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to measure binding constants of proteins binding to DNA reliably, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This change in bead motion makes it possible to measure the DNA-binding properties of proteins. We use the bacterial protein integration host factor (IHF) and the archaeal histone HMfA as examples to show how specific binding to DNA can be measured. Moreover, we show how the end-to-end distance can provide structural insights into protein-DNA binding.

Rok from B. subtilis: Bridging genome structure and transcription regulation.

Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface.

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into 'hypernucleosome' particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

Specific DNA binding of archaeal histones HMfA and HMfB.

In archaea, histones play a role in genome compaction and are involved in transcription regulation. Whereas archaeal histones bind DNA without sequence specificity, they bind preferentially to DNA containing repeats of alternating A/T and G/C motifs. These motifs are also present on the artificial sequence "Clone20," a high-affinity model sequence for binding of the histones from Methanothermus fervidus. Here, we investigate the binding of HMfA and HMfB to Clone20 DNA. We show that specific binding at low protein concentrations (<30 nM) yields a modest level of DNA compaction, attributed to tetrameric nucleosome formation, whereas nonspecific binding strongly compacts DNA. We also demonstrate that histones impaired in hypernucleosome formation are still able to recognize the Clone20 sequence. Histone tetramers indeed exhibit a higher binding affinity for Clone20 than nonspecific DNA. Our results indicate that a high-affinity DNA sequence does not act as a nucleation site, but is bound by a tetramer which we propose is geometrically different from the hypernucleosome. Such a mode of histone binding might permit sequence-driven modulation of hypernucleosome size. These findings might be extrapolated to histone variants that do not form hypernucleosomes. Versatile binding modes of histones could provide a platform for functional interplay between genome compaction and transcription.

The B. subtilis Rok protein is an atypical H-NS-like protein irresponsive to physico-chemical cues.

Nucleoid-associated proteins (NAPs) play a central role in chromosome organization and environment-responsive transcription regulation. The Bacillus subtilis-encoded NAP Rok binds preferentially AT-rich regions of the genome, which often contain genes of foreign origin that are silenced by Rok binding. Additionally, Rok plays a role in chromosome architecture by binding in genomic clusters and promoting chromosomal loop formation. Based on this, Rok was proposed to be a functional homolog of E. coli H-NS. However, it is largely unclear how Rok binds DNA, how it represses transcription and whether Rok mediates environment-responsive gene regulation. Here, we investigated Rok's DNA binding properties and the effects of physico-chemical conditions thereon. We demonstrate that Rok is a DNA bridging protein similar to prototypical H-NS-like proteins. However, unlike these proteins, the DNA bridging ability of Rok is not affected by changes in physico-chemical conditions. The DNA binding properties of the Rok interaction partner sRok are affected by salt concentration. This suggests that in a minority of Bacillus strains Rok activity can be modulated by sRok, and thus respond indirectly to environmental stimuli. Despite several functional similarities, the absence of a direct response to physico-chemical changes establishes Rok as disparate member of the H-NS family.

System-Wide Analysis of the GATC-Binding Nucleoid-Associated Protein Gbn and Its Impact on Streptomyces Development.

Bacterial chromosome structure is, to a great extent, organized by a diverse group of proteins collectively referred to as nucleoid-associated proteins (NAPs). Many NAPs have been well studied in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of Actinobacteria NAPs and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein, which is expressed in particular during sporulation, was designated Gbn for GATC-binding NAP. Deletion of gbn led to alterations in development and antibiotic production in Streptomyces coelicolor. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2,800 binding regions, encompassing around 3,600 GATCWT motifs. This amounts to 55% of all such sequences in the S. coelicolor genome. DNA binding of Gbn in vitro minimally changes DNA conformation, suggesting a modest role in chromosome organization only, in addition to a gene regulatory role. Transcriptomics analysis showed that Gbn binding generally leads to reduced gene expression. The DNA binding profiles were nearly identical between vegetative and aerial growth. Exceptions are SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognizes the rare leucine codon CUA, respectively, which nearly exclusively bound during vegetative growth. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes. IMPORTANCE A large part of the chemical space of bioactive natural products is derived from Actinobacteria. Many of the biosynthetic gene clusters for these compounds are cryptic; in others words, they are expressed in nature but not in the laboratory. Understanding the global regulatory networks that control gene expression is key to the development of approaches to activate this biosynthetic potential. Chromosome structure has a major impact on the control of gene expression in eukaryotes. In bacteria, the organization of chromosome structure is mediated by multiple factors, including macromolecular biophysics processes, biological processes, and, more importantly, a diverse group of proteins referred to collectively as nucleoid-associated proteins (NAPs). We here present the discovery of a novel and extremely pleiotropic NAP, which we refer to as Gbn. Gbn is an Actinobacteria-specific protein that binds to GATC sequences, with a subtle but broad effect on global gene expression, especially during the late developmental stage. The discovery of Gbn is a new step toward better understanding of how gene expression and chromosome structure are governed in antibiotic-producing streptomycetes.

Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity.

Horizontal gene transfer facilitates dissemination of favourable traits among bacteria. However, foreign DNA can also reduce host fitness: incoming sequences with a higher AT content than the host genome can misdirect transcription. Xenogeneic silencing proteins counteract this by modulating RNA polymerase binding. In this work, we compare xenogeneic silencing strategies of two distantly related model organisms: Escherichia coli and Bacillus subtilis. In E. coli, silencing is mediated by the H-NS protein that binds extensively across horizontally acquired genes. This prevents spurious non-coding transcription, mostly intragenic in origin. By contrast, binding of the B. subtilis Rok protein is more targeted and mostly silences expression of functional mRNAs. The difference reflects contrasting transcriptional promiscuity in E. coli and B. subtilis, largely attributable to housekeeping RNA polymerase σ factors. Thus, whilst RNA polymerase specificity is key to the xenogeneic silencing strategy of B. subtilis, transcriptional promiscuity must be overcome to silence horizontally acquired DNA in E. coli.

Novel anti-repression mechanism of H-NS proteins by a phage protein.

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins.

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.