Protected region upstream and limited nucleosomal positioning downstream of the transcription initiation region of yeast 35S ribosomal gene

Abstract

To locate nucleosome positions around the transcription initiation site of 35S rDNA in yeast, single and double cleavage site data for staphylococcal nuclease and single cleavage site data for DNase I were obtained by methods involving some modifications of the usual indirect end-labeling procedure. Results show that immediately upstream of the initiation site there is an approximately 225 base pair (bp) stretch of chromatin which is quite resistant to nuclease digestion. The nucleosomal domain begins on the coding sequences, at the downstream end of the resistant region, with the major locus 30 bp and a minor locus 5 bp downstream of the initiation site in growing cells. The location of the major locus is corroborated by analysis within the coding sequences, although results within the gene are less convincing because of a strong similarity of chromatin and naked DNA digestion profiles. The staphylococcal nuclease cleavage maps were tested by their ability to reproduce chromatin digest profiles and to explain the presence or absence, depending on which parts of the region were used as probes, of specific bands within the pattern. The site of the major upstream locus of the nucleosomal domain differs in growing and stationary cells. This shift changes the accessibility of approximately 25 bp of DNA and may be associated with the expression of these genes.

Organization of theGAL1–GAL10intergeaic control region chromatin

“…In both inactive and active chromatin digests, the UAS region chromatin seems to be digested somewhat faster than the surrounding chromatin, as judged by comparison of the relative yields of mono-versus higher molecular weight DNA in the hinfl compared to p or 6 profiles (Figure 4B, trs. 2 vs. 1,3; 5 vs. 4,6). This is consistent with the cleavage site data shown in Figure 3.…”

“…Intracore bands #9 and #11 are located in the profiles. Probably because 6 is so small, it was not possible from a normally loaded gel to get a profile in which the signal was strong enough or the background low enough to show up properly in a photograph, so for track 4 an -200 bp probe from the region immediately downstream of 6 was used instead (6*).…”

“…However, the structural causes for these sensitivities and details of the chromatin structure of any upstream (control) region remain largely unknown. Suggestions about the structure of a ribosomal upstream region have been made (5,6), but the presence of inactive genes in this repeated set makes the structural information ambiguous. The structure in gene upstream regions is of interest because these regions contain DNA sequences thought to be involved in controlling gene expression and the chromatin structure may be an aspect of that control.…”