Two related regulatory sequences are required for maximal induction of Saccharomyces cerevisiae his3 transcription.

Abstract

In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extending to -94 or beyond do not produce detectable levels of his3 mRNA. Small internal deletions that only remove the proximal regulatory sequence and a 1-base-pair deletion of the thymine residue at -99 abolish induction, but do not affect the basal level of transcription. These results indicate that the proximal copy between -99 and -94 is absolutely required for his3 induction, whereas the copy between -142 and -137 is required only for the maximal level of induction and is inactive by itself. From these and other observations, we suggest the possibility that these related regulatory sequences may be targets for two distinct proteins.

Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the Entire Gene

“…This problem was circumvented by constructing a strain with a mutated Gcn4p binding site and expressing both SNF2-FLAG and GCN4-myc. The mutation was a single-base deletion (of the first T in the TGACTC sequence recognized by Gcn4p; the his3-142 mutation [34]). In order to introduce this mutation into the chromosomal HIS3 gene, it was necessary to insert a selection marker (ADE2) into the HIS3 ORF, rendering the strain unable to grow in the absence of histidine.…”

“…(F) Gcn4p does not recruit significant amounts of SWI/SNF to the HIS3 promoter. Strains with or without a point deletion in the binding site for Gcn4p in the HIS3 promoter (the his3-142 mutation [34]) were used. These strains expressed both Gcn4-myc and Snf2-FLAG.…”

Preferential Accessibility of the Yeast his3 Promoter Is Determined by a General Property of the DNA Sequence, Not by Specific Elements

“…Detailed mutational analysis of the his3 promoter region has identified the following promoter elements: initiator elements that specify the ϩ1 and ϩ13 mRNA start sites (2); a consensus TATA element (nucleotides Ϫ45 to Ϫ40), T R , that is responsible for ϩ13 transcription (3); a collection of nonconsensus TATA elements (nucleotides Ϫ80 to Ϫ53), T C , that is responsible for ϩ1 initiation (14, 28); a Gcn4 binding site located between nucleotides Ϫ100 and Ϫ91 and an adjacent tract of 9 dA-dT residues (11); a poly(dA-dT) element (nucleotides Ϫ130 to Ϫ115) that is important for Gcn4-independent transcription of his3 as well as the divergently transcribed pet56 (15,40); and a poorly characterized sequence around nucleotide Ϫ140 which makes a minor contribution to maximal induced levels of transcription (44). In addition, there is a nonconsensus TATA element (nucleotide Ϫ150) that is responsible for pet56 transcription, which is initiated in the direction opposite from a position 191 bp upstream of his3 ϩ1 (40).…”

RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.

“…Samples containing equal amounts of the large rRNAs were subjected to electrophoresis in 1% formaldehyde-agarose gels, transferred to nitrocellulose, and hybridized with nick-translated DNA probes (27,29). The plasmids that were used as probes were pY2085 (a pUC18 plasmid containing a 7.1-kb HindIll DNA fragment of RPBI [42]), a pY2315 (a pGEM3 plasmid containing a 5.3-kb EcoRI-HindIII DNA fragment bearing LYS2 [4]), pY2317 (a pGEM3 plasmid containing a 1.65-kb BamHIHindIll DNA fragment bearing ACT] [32]), pY2384 (a pUC8 plasmid containing a 2.6-kb XhoI-SalI DNA fragment bearing DEDI [37]), and pYG100 (a pBR322 plasmid bearing the 8-kb BamHI-HindIII DNA fragment bearing SSAI [20]). After hybridization, the blots were washed four times at 550C in 0.1 x SSPE (27)-0.1% sodium dodecyl sulfate (SDS).…”