Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase.

Here we investigated the refolding of Bacillus subtilis 6S-1 RNA and its release from σA-RNA polymerase (σA-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σA-RNAP affinity. This indicates that the RNA's extended helical arms including the '-35'-like region are not required for basic 6S-1 RNA functionality. The role of the 'central bubble collapse helix' (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σA-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5'-part of the CB (5'-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σA-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3'-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA's gel mobility and interfered with σA-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5'-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.

Silanol-based surfactants: synthetic access and properties of an innovative class of environmentally benign detergents.

Herein, environmentally friendly surfactants based on new silanols as substitutes for the isoelectronic phosphonates were explored. Surface tensions of aqueous solutions are significantly reduced, particularly with those silanols that feature a high ratio of organic moiety to silanol. Besides their use as surfactants, their potential as coating agents for hydrophilic oxide surfaces was investigated for the example of glass substrates. In the solid-state sheet structures with silanol, double layers are present, in which the sheet spacing varies with the alkyl-chain length.

Segrosome assembly at the pliable parH centromere.

The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere. Here we demonstrate that the distinctive parH site (∼100-bp) consists of an array of degenerate tetramer boxes interspersed by AT-rich spacers. Although numerous consecutive AT-steps are suggestive of inherent curvature, parH lacks an intrinsic bend. Sequential deletion of parH tetramers progressively reduced centromere function. Nevertheless, the variant subsites could be rearranged in different geometries that accommodated centromere activity effectively revealing that the site is highly elastic in vivo. ParG cooperatively coated parH: proper centromere binding necessitated the protein's N-terminal flexible tails which modulate the centromere binding affinity of ParG. Interaction of the ParG ribbon-helix-helix domain with major groove bases in the tetramer boxes likely provides direct readout of the centromere. In contrast, the AT-rich spacers may be implicated in indirect readout that mediates cooperativity between ParG dimers assembled on adjacent boxes. ParF alone does not bind parH but instead loads into the segrosome interactively with ParG, thereby subtly altering centromere conformation. Assembly of ParF into the complex requires the N-terminal flexible tails in ParG that are contacted by ParF.

Filamentous biopolymers on surfaces: atomic force microscopy images compared with Brownian dynamics simulation of filament deposition.

Nanomechanical properties of filamentous biopolymers, such as the persistence length, may be determined from two-dimensional images of molecules immobilized on surfaces. For a single filament in solution, two principal adsorption scenarios are possible. Both scenarios depend primarily on the interaction strength between the filament and the support: i) For interactions in the range of the thermal energy, the filament can freely equilibrate on the surface during adsorption; ii) For interactions much stronger than the thermal energy, the filament will be captured by the surface without having equilibrated. Such a 'trapping' mechanism leads to more condensed filament images and hence to a smaller value for the apparent persistence length. To understand the capture mechanism in more detail we have performed Brownian dynamics simulations of relatively short filaments by taking the two extreme scenarios into account. We then compared these 'ideal' adsorption scenarios with observed images of immobilized vimentin intermediate filaments on different surfaces. We found a good agreement between the contours of the deposited vimentin filaments on mica ('ideal' trapping) and on glass ('ideal' equilibrated) with our simulations. Based on these data, we have developed a strategy to reliably extract the persistence length of short worm-like chain fragments or network forming filaments with unknown polymer-surface interactions.

Sequence-specific physical properties of African green monkey alpha-satellite DNA contribute to centromeric heterochromatin formation.

Satellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cell division. Structural properties of alpha-satellite DNA (AS) from African green monkey (AGM) were studied. Atomic force microscopy imaging showed smaller end-to-end distances of AS fragments than would be expected for the persistence length of random sequence DNA. The apparent persistence length of the AS was determined as 35nm. Gel-electrophoresis indicated only a weak contribution of intrinsic curvature to the DNA conformations suggesting an additional contribution of an elevated bending flexibility to the reduced end-to-end distances. Next, the force-extension behavior of the naked AS and in complex with nucleosomes was studied using optical tweezers. The naked AS showed a reduced overstretching transition force (-18% the value determined for random DNA) and higher forces required to straighten the DNA. Finally, reconstituted AS nucleosomes disrupted at significantly higher forces as compared with random DNA nucleosomes which is probably due to structural properties of the AS which stabilize the nucleosomes. The data support that the AS plays a role in the formation of centromeric heterochromatin due to specific structural properties and suggest that a relatively higher mechanical stability of nucleosomes is important in AGM-AS chromatin.

Spontaneous access to DNA target sites in folded chromatin fibers.

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from approximately 3-fold decreases to approximately 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as approximately 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.

Centromere anatomy in the multidrug-resistant pathogen Enterococcus faecium.

Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.

Organisation of nucleosomal arrays reconstituted with repetitive African green monkey alpha-satellite DNA as analysed by atomic force microscopy.

Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45-50%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats.

Trinucleosome compaction studied by fluorescence energy transfer and scanning force microscopy.

The effect of the salt concentration, linker histone H1, and histone acetylation on the structure of trinucleosomes reconstituted on a 608 bp DNA containing one centered nucleosome positioning signal was studied. Fluorescence resonance energy transfer (FRET) in solution and scanning force microscopy (SFM) measurements in liquid were done on the same samples. The distance between the DNA ends decreases under the effect of an increasing salt concentration and also by the incorporation of the H1 linker histone. A decrease of internucleosomal center-to-center (cc) distances by H1 was observed that was limited to a minimal value of about 20 nm. The distribution of the angle formed between consecutive nucleosomes was narrowed by H1. The effect of acetylation of all histones leads to decompaction, measured as an increased distance between the DNA ends, and also increased the internucleosomal distances. Selective acetylation of histone H4, however, compacts the structure as measured by FRET.

DNA-loop formation on nucleosomes shown by in situ scanning force microscopy of supercoiled DNA.

The flexibility of the chromatin structure, necessary for the processing of the genomic DNA, is controlled by a number of factors where flexibility and mobility of the nucleosomes is essential. Here, the influence of DNA supercoiling on the structure of single nucleosomes is investigated. Circular supercoiled plasmid DNA sub-saturated with histones was visualized by scanning force microscopy (SFM) in aqueous solution. SFM-imaging compared with topological analysis indicates instability of nucleosomes when the salt concentration is raised from 10 mM to 100 mM NaCl. Nucleosomes were observed after the deposition to the used scanning surface, i.e. mica coated with polylysine. On the images, the nucleosomes appear with a high probability in end-loops near the apices of the superhelices. In 100 mM NaCl but not in 10 mM NaCl, a significant number of complexes present the nucleosomes on superhelical crossings mainly located adjacent to an end-loop. The morphology of these structures and statistical analysis suggest that DNA loops were formed on the histone octamers, where the loop size distribution shows a pronounced peak at 50 nm. Recently, the formation and diffusion of loops on octamers has been discussed as a mechanism of translocations of nucleosomes along DNA. The presented data likely confirm the occurrence of loops, which may be stabilized by supercoiling. Analysis of the structure of regular nucleosomes not located on crossings indicates that reducing the salt concentration leads to more conformations, where DNA is partially unwrapped from the distal ends of the octamer.

Polylysine-coated mica can be used to observe systematic changes in the supercoiled DNA conformation by scanning force microscopy in solution.

The conformations of supercoiled (sc) DNA and linear DNA bound to polylysine (PL)-coated mica were investigated by scanning force microscopy (SFM) in solution. From the polymer statistical analysis of linear DNA, we could distinguish between re-arrangements or trapping of the DNA on the surface. Conditions of re-arrangements to an almost equilibrated state can be achieved at appropriate PL surface concentrations. We could show that the ability of re-arrangements depends on the salt concentration of the adsorption/imaging buffer. Comparing the statistical analysis of the linear DNA with SFM images of scDNA suggested that irregular scDNA conformations are formed under conditions of trapping, whereas plectonemic structures are favoured under conditions of surface re-arrangements. Salt-dependent changes in the scDNA conformation over the range of 10-100 mM NaCl, as characterised by the parameters writhe and the superhelix radius r, are observable only under conditions that enable surface re-arrangements. The measured values of writhe suggest that the scDNA loses approximately one-half of the supercoils during the binding to the surface. At the same time r increases systematically with decreasing writhe, thus the scDNA topology remains determined by the constraints on supercoiling during the binding to PL-coated mica.

Kinetics of site-site interactions in supercoiled DNA with bent sequences.

A curved DNA segment is known to adopt a preferred end loop localization in superhelical (sc) DNA and thus may organize the overall conformation of the molecule. Through this process it influences the probability of site juxtaposition. We addressed the effect of a curvature on site-site interactions quantitatively by measuring the kinetics of cross-linking of two biotinylated positions in scDNA by streptavidin. The DNA was biotinylated at either symmetric or asymmetric positions with respect to a curved insert via triplex-forming oligonucleotides (TFOs) modified with biotin. We used a quench-flow device to mix the DNA with the protein and scanning force microscopy to quantify the reaction products. As a measure of the interaction probability, rate constants of cross-linking and local concentrations j(M) of one biotinylated site in the vicinity of the other were determined and compared to Monte Carlo simulations for corresponding DNAs. In good agreement with the simulations, a j(M) value of 1.74 microM between two sites 500bp apart was measured for an scDNA without curvature. When a curvature was centered between the sites, the interaction probability increased about twofold over the DNA without curvature, significantly less than expected from the simulations. However, the relative differences of the interaction probabilities due to varied biotin positions with respect to the curvature agreed quantitatively with the theory.