In yeast cells, the Rtf1 and Paf1 components of the Paf1 transcriptional elongation complex are important for recruitment of Set1, the histone H3-lysine 4 (H3-Lys 4 ) methylase, to a highly localized domain at the 5 portion of active mRNA coding regions. Here, we show that Rtf1 is essential for global methylation of H3-Lys 4 and H3-Lys 79 , but not H3-Lys 36 . This role of Rtf1 resembles that of Rad6, which mediates ubiquitination of histone H2B at lysine 123. Indeed, Rtf1 is required for H2B ubiquitination, suggesting that its effects on H3-Lys 4 and H3-Lys 79 methylation are an indirect consequence of its effect on H2B ubiquitination. Rtf1 is important for telomeric silencing, with loss of H3-Lys 4 and H3-Lys 79 methylation synergistically reducing Sir2 association with telomeric DNA. Dot1, the H3-Lys 79 methylase, associates with transcriptionally active genes, but unlike the association of Set1 and Set2 (the H3-Lys 36 methylase), this association is largely independent of Rtf1. We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor.In a variety of eukaryotic organisms, histone H3 is methylated at lysines 4, 36, and 79 (respectively H3-Lys 4 , H3-Lys 36 , H3-Lys 79 ). In the budding yeast Saccharomyces cerevisiae, H3-Lys 4 is methylated by Set1 (1-4), H3-Lys 36 is methylated by Set2 (5), and H3-Lys 79 is methylated by Dot1 (6 -8). Unexpectedly, genome-wide methylation of H3-Lys 4 and H3-Lys 79 depends on Rad6, an enzyme that mono-ubiquitinates lysine 123 of histone H2B (9 -12). H3-Lys 4 and H3-Lys 79 methylation also depends on Bre1, an E3 ubiquitin ligase that is required for substrate selection of Rad6 (13,14). The relationship between ubiquitination of H2B-Lys 123 and methylation of H3 at lysines 4 and 79 is unidirectional, as the loss of H3 methylation does not influence H2B ubiquitination.Set1 and Dot1 are important for heterochromatic silencing (1,3,6,7,15,16), whereas Set2 does not appear to play a role in this process. Dot1-mediated methylation and H3-Lys 79 itself are important for association of Sir silencing proteins at telomeres, with the effects being more dramatic at telomeredistal regions than at telomere-proximal regions (6, 7 (18) is extremely low at heterochromatic loci, and H3-Lys 79 methylation occurs at high levels in bulk chromatin (6). These observations suggest that Sir proteins preferentially associate with nucleosomes that are unmethylated at H3-Lys 79 (6, 18) and that Sir proteins block the ability of Dot1 to methylate H3-Lys 79 (18). In addition to its role in heterochromatin silencing, Set1 has an important role in transcriptional elongation. Set1 mediates both di-and trimethylation of H3-Lys 4 , and trimethylation correlates with transcriptional activity (19). Set1 is specifically recruited by elongating RNA polymerase II (pol II) 1 to a highly localized domain at the 5Ј region of actively transcribed genes, thereby generating a highly localized domain of trimethylated H3-Lys 4 (20)....

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Background: Recent advances in synthesizing valuable chemicals such as organic acids from low-cost renewable biomass through microbial fermentation have attracted great attention. However, the toxicity of organic acids presents a key challenge to the development of an economically viable fermentation process. Therefore, a platform organism that not only produces organic acids but also tolerates the associated toxicity is highly desirable. Results: Here we report the discovery, characterization, and engineering of a yeast strain, Issatchenkia orientalis SD108, that is tolerant to low pH and high concentration of organic acids. This strain demonstrated a higher tolerance compared to I. orientalis ATCC 24210 and Classic Distiller's Turbo yeast. In order to explore SD108 as a potential platform organism for organic acid production, we determined its draft genome sequence and use the sequencing information to guide pathway design. As proof of concept, an engineered four-gene expression cassette related to the reductive TCA cycle was assembled and integrated into the genome of a uracil auxotroph of SD108. The resulting strain was able to produce succinic acid with a titer of 11.63 g/L, yield of 0.12 g/g, and productivity of 0.11 g/L · h in batch cultures using shake flasks.

H-NS inhibits transcription by forming repressing nucleoprotein complexes next to promoters. We investigated repression by binding of H-NS within the transcription unit using the bgl and proU operons. Repression of both operons requires a downstream regulatory element (DRE) in addition to an upstream element (URE). In bgl, H-NS binds to a region located between 600 to 700 bp downstream of the transcription start site, whereas in proU the DRE extends up to position ؉270. We show that binding of H-NS to the bgl-DRE inhibits transcription initiation at a step before open complex formation, as shown before for proU. This was shown by determining the occupancy of the bgl transcription unit by RNA polymerases, expression analysis of bgl and proU reporter constructs, and chloroacetaldehyde footprinting of RNA polymerase promoter complexes. The chloroacetaldehyde footprinting also revealed that RNA polymerase is "poised" at the osmoregulated 70-dependent proU promoter at low osmolarity, whereas at high osmolarity poising of RNA polymerase and repression by H-NS are reduced. Furthermore, repression by H-NS via the URE and DRE is synergistic, and the efficiency of repression by H-NS via the DRE inversely correlates with the promoter activity. Repression is high for a promoter of low activity, whereas it is low for a strong promoter. Inefficient repression of strong promoters by H-NS via a DRE may account for high induction levels of proU at high osmolarity and for bgl upon disruption of the URE.

Summary The silent bgl operon of Escherichia coli is activated by spontaneous mutations that derepress its promoter. In addition, expression depends on specific transcriptional antitermination within the operon by the antiterminator protein BglG. Here, we show that BglG‐mediated antitermination limits expression of the bgl operon when the cellular transcription rate is low. The expression levels of chromosomally encoded activated bgl operon alleles are low but increase significantly when BglG protein is provided in trans or when the expression is rendered inde‐pendent of BglG‐mediated antitermination by mutation of the terminator. Plasmid‐encoded activated bgl operon alleles are expressed at high levels. Moreover, a moderate (threefold) further increase in the transcription rate of chromosomally encoded activated bgl operon alleles in an rpoS mutant can result in high (up to 50‐fold increased) expression levels. These data show that the expression of the bgl operon does not correlate linearly with its cellular transcription rate. Moderate differences in the transcription initiation rate are amplified post‐transcriptionally into large changes in the expression level of the operon by the requirement of a threshold for BglG‐mediated antitermination. Implications for bgl operon regulation by global regulators H‐NS, RpoS and others are discussed.