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Studying transcription machinery assembly in vitro is challenging because of long intrinsically disordered regions present within the multi-modular transcription factors. One example is alcohol dehydrogenase repressor 1 (Adr1p) from fermenting yeast, responsible for the metabolic switch from glucose to ethanol. The role of each individual transcription activation domain (TAD) has been previously studied, but their interplay and their roles in enhancing the stability of the protein is not known. In this work, we designed five unique miniAdr1 constructs containing either TADs I-II-III or TAD I and III, connected by linkers of different sizes and compositions. We demonstrated that miniAdr1-BL, containing only PAR-TAD I+III with a basic linker (BL), binds the cognate DNA sequence, located in the promoter of the ADH2 (alcohol dehydrogenase 2) gene, and is necessary to stabilize the heterologous expression. In fact, we found that the sequence of the linker between TAD I and III affected the solubility of free miniAdr1 proteins, as well as the stability of their complexes with DNA. miniAdr1-BL is the stable unit able to recognize ADH2 in vitro , and hence it is a promising tool for future studies on nucleosomal DNA binding and transcription machinery assembly in vitro ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The effects of the rotational information of DNA in determining the in vivo localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5 S rRNA repeat gene. The distribution of the phased series of flexibility signals present in this DNA has been altered by inserting in its centre a 25 bp tract. The effects of such alteration on the in vivo distribution of the helically phased, alternatively located ncps have been determined relative to a reference 21 bp insertion mutant. The results show that the answers provided in vitro and in vivo by the yeast 5 S rRNA gene sequence to specific modifications of the DNA rotational frame are similar, thus pointing to the relevance of DNA rotational information in vivo.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

A simple no-background assay was developed for high-resolution in vivo analysis of yeast chromatin. When applied to Saccharomyces cerevisiae 5S rRNA genes (5S rDNA), this analysis shows that nucleosomes completely cover this chromosomal region, occupying alternative positions characterized by a unique helical phase. This supports the notion that sequence-intrinsic rotational signals are the major determinant of nucleosome localization. Nucleosomal core particles reconstituted in vitro occupy the same positions and have the same helically phased distribution observed in vivo, as determined by mapping of exonuclease III-resistant borders, mapping by restriction cleavages, and by DNase I and hydroxyl-radical digestion patterns.