Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed lambda-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32 degrees C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

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We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the longscale structure of the bacterial nucleoid.T he genetic material in bacterial cells is organized in a structure called the nucleoid (1-3). In Escherichia coli, this nucleoprotein complex consists of a single circular DNA molecule 4.7 million bp long, RNA, and a large variety of bound proteins. Among these, about 10 so-called histone-like proteins, including HU, integration host factor (IHF), and H-NS (1-3), shape the short-scale structure of the nucleoid by bending DNA locally on binding. These proteins therefore play an important role in compacting the DNA molecule, in addition to other factors such as supercoiling (4), macromolecular crowding (5), and osmotic effects (6).The level of nucleoid-associated proteins changes as a function of bacterial growth. For example, the level of IHF was shown to increase on entry to the stationary phase of growth, becoming one of the major histone-like proteins in the cell (7-10). By binding to specific DNA sites, IHF participates in forming higher-order DNA structures required for replication, sitespecific recombination, phage packaging, and regulation of transcription initiation (8). IHF can also bind to DNA nonspecifically and can be substituted by HU. In fact, IHF and HU possess similar overall structures and share several regions of conserved homologies (11,12).Very little is known about the structural modifications on DNA induced by histone-like proteins in nucleoid formation. In particular, the degree of compaction induced by each of these proteins has not been quantified, and information about the large-scale structure of nucleoprotein complexes is scarce. This deficiency stems in part from the fact that classical techniques used in molecular biology are designed for studying relatively strong DNA-binding sites and cannot appropriately assess the contribution of nonspecific low-affinity interactions. Furthermore, analyses based on electron micr...

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We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wideswath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation.Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo. INTRODUCTIONThe regulation of gene expression is a process central to all biological life-forms. It is a process thought to be mediated largely by proteins, which interact with either chromatin or its RNA product.The best-known form of regulation is mediated by transcription factors, which control RNA levels by their sequence-specific interaction with DNA. Post transcriptional regulation based on protein-RNA interactions, however, is quite different, due to the nature of RNA. Unlike DNA which is a long, chromatinized, replicated, and for the most part exists as a double stranded molecule, RNA is a short, transient (i.e. constantly manufactured and degraded), exists in multiple copies, has particular modifications (1, 2), and folds into functional secondary and tertiary structures. RNA structure is