H-NS mediated repression of the Escherichia coli bgl and proU operons

The histone-like nucleoid structuring protein H-NS is important in the organization of the bacterial chromosome and in global gene regulation in response to environmental stimuli and stress conditions. In Enterobacteriaceae such as Escherichia coli H-NS represses ~5 percent of all genes. Repression by H-NS is presumably mediated by binding of H-NS next to a promoter, and the formation of extended nucleoprotein complex, which inhibits transcription initiation. Although the specificity of binding of H-NS to DNA is low (it binds weakly specific to AT-rich curved DNA), some loci are very specifically repressed by H-NS including the E. coli bgl and proU operons. In both of these systems, upstream and downstream regulatory elements are required for efficient repression. In bgl H-NS binds 600 to 700 bp downstream to the promoter and in proU it binds 150 to 300 bp downstream. The analysis done here suggests that repression of proU and bgl by binding of H-NS to upstream and downstream regulatory elements is cooperative. Furthermore, it was shown that in the absence of the upstream regulatory element (URE), repression by H-NS binding to the downstream regulatory element (DRE) depends on the transcription rate. Termination factor Rho and co-transcriptional translation, which both modulate the transcription rate, were shown to also affect repression by H-NS via the DRE. Further experiments excluded, that H-NS acts as a roadblock to the transcribing RNA polymerase. In the bgl operon H-NS represses transcription elongation merely 2-fold and in proU it has no effect on elongation. These experiments include CAA-footprinting of stalled RNA polymerase transcription elongation complexes, Northern analysis, and a dual reporter gene system with the bgl and proU DRE, respectively, inserted in between uidA (?-glucuronidase) and lacZ (?galactosidase). In addition, the analysis of structural components in bgl revealed an intrinsic transcription pause site located in between the promoter and the bgl-DRE. However, the deletion of the pause did not affect repression. Additional deletion analyses suggest that the DNA segment upstream of the bgl-DRE is important for repression. The data shown here and ongoing experiments suggest that binding of H-NS to the DRE inhibits transcription initiation at the bgl and proU promoter, respectively. Possibly, H-NS bound to the DRE traps a DNA segment located upstream of the promoter resulting in DNA looping and repression of transcription initiation. Furthermore, the present work highlights the significance of the transcription rate and the process of transcription elongation in the modulation of H-NS mediated repression. Presumably, an increase in the transcription rate de-stabilizes the repressing complex formed by H-NS and thus causes full expression

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these \u27proto-chromatosomes\u27 are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1

A polar barrier to transcription can be circumvented by remodeler-induced nucleosome translocation

Many eukaryotic genes are regulated at the level of transcript elongation. Nucleosomes are likely targets for this regulation. Previously, we have shown that nucleosomes formed on very strong positioning sequences (601 and 603), present a high, orientation-dependent barrier to transcription by RNA polymerase II in vitro. The existence of this polar barrier correlates with the interaction of a 16-bp polar barrier signal (PBS) with the promoter-distal histone H3–H4 dimer. Here, we show that the polar barrier is relieved by ISW2, an ATP-dependent chromatin remodeler, which translocates the nucleosome over a short distance, such that the PBS no longer interacts with the distal H3–H4 dimer, although it remains within the nucleosome. In vivo, insertion of the 603 positioning sequence into the yeast CUP1 gene results in a modest reduction in transcription, but this reduction is orientation-independent, indicating that the polar barrier can be circumvented. However, the 603-nucleosome is present at the expected position in only a small fraction of cells. Thus, the polar barrier is probably non-functional in vivo because the nucleosome is not positioned appropriately, presumably due to nucleosome sliding activities. We suggest that interactions between PBSs and chromatin remodelers might have significant regulatory potential

tRNA biology in the omics era: Stress signalling dynamics and cancer progression.

Recent years have seen a burst in the number of studies investigating tRNA biology. With the transition from a gene-centred to a genome-centred perspective, tRNAs and other RNA polymerase III transcripts surfaced as active regulators of normal cell physiology and disease. Novel strategies removing some of the hurdles that prevent quantitative tRNA profiling revealed that the differential exploitation of the tRNA pool critically affects the ability of the cell to balance protein homeostasis during normal and stress conditions. Furthermore, growing evidence indicates that the adaptation of tRNA synthesis to cellular dynamics can influence translation and mRNA stability to drive carcinogenesis and other pathological disorders. This review explores the contribution given by genomics, transcriptomics and epitranscriptomics to the discovery of emerging tRNA functions, and gives insights into some of the technical challenges that still limit our understanding of the RNA polymerase III transcriptional machinery

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli

Function of TFIIIC, RNA polymerase III initiation factor, in activation and repression of tRNA gene transcription

Transcription of transfer RNA genes by RNA polymerase III (Pol III) is controlled by general factors, TFIIIB and TFIIIC, and a negative regulator, Maf1. Here we report the interplay between TFIIIC and Maf1 in controlling Pol III activity upon the physiological switch of yeast from fermentation to respiration. TFIIIC directly competes with Pol III for chromatin occupancy as demonstrated by inversely correlated tDNA binding. The association of TFIIIC with tDNA was stronger under unfavorable respiratory conditions and in the presence of Maf1. Induction of tDNA transcription by glucose-activated protein kinase A (PKA) was correlated with the down-regulation of TFIIIC occupancy on tDNA. The conditions that activate the PKA signaling pathway promoted the binding of TFIIIB subunits, Brf1 and Bdp1, with tDNA, but decreased their interaction with TFIIIC. Association of Brf1 and Bdp1 with TFIIIC was much stronger under repressive conditions, potentially restricting TFIIIB recruitment to tDNA and preventing Pol III recruitment. Altogether, we propose a model in which, depending on growth conditions, TFIIIC promotes activation or repression of tDNA transcription

Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria

During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes