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

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

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

Data_Sheet_1_Specific DNA binding of archaeal histones HMfA and HMfB.PDF

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.</p

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

Abstract 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

Mechanical and structural properties of archaeal hypernucleosomes

Macromolecular Biochemistr