Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein.

Abstract

General regulatory factor I (GRFI) is a yeast protein that binds in vitro to specific DNA sequences at diverse genetic elements. A strategy was pursued to test whether GRFI functions in vivo at the sequences bound by the factor in vitro. Matches to a consensus sequence for GRFI binding were found in a variety of locations: upstream activating sequences (UASs), silencers, telomeres, and transcribed regions. All occurrences of the consensus sequence bound both crude and purified GRFI in vitro. All binding sites for GRFI, regardless of origin, provided UAS function in test plasmids. Also, GRFI binding sites specifically stimulated transcription in a yeast in vitro system, indicating that GRFI can function as a positive transcription factor. The stimulatory effect of GRFI binding sites at UASs for the PYKI and ENO] genes is significantly enhanced by flanking DNA elements. By contrast, regulatory sequences that flank the GRFI binding site at HMR E convert this region to a transcriptional silencer.

Activation of gastrin gene transcription in islet cells by a RAP1‐like cis‐acting promoter element

“…la). Furthermore, a good match to the consensus recognition sequence for the yeast DNA binding protein RAP1, A(A/ C)ACCCANN(C/A)(C/A)Y(C/A), [10,22,27] was observed in this conserved promoter region of the gastrin gene (Fig. lb).…”

“…RAP1, also known as GRF1 or TURF [4,5] is a highly abundant multifunctional DNA binding protein which effects both transcriptional activation and silencing in yeast. The RAP1 binding site at the HMR silent mating-type locus is required for full repression of transcription [6][7][8][9][10]. RAPI binding sites are also found upstream of a large number of genes including ribosomal and glycolytic enzyme genes activating gene transcription [11][12][13][14].…”

“…2a, lane 6). This mutation has been shown to prevent RAP1 binding in yeast promoters [8,10]. The nucleotide residues int he rGRD DNA element necessary for RAPI binding were determined by methylation interference analysis.…”

“…RAPI binding sites are also found upstream of a large number of genes including ribosomal and glycolytic enzyme genes activating gene transcription [11][12][13][14]. RAP1 also binds to ARSs (autonomously replicating sequences) and telomeres of yeast chromosomes to regulate cell division [10,15]. Since RAP1 regulates many biological processes common to yeast and mammalian cells, homologs of RAP1 with conserved DNA binding domains could regulate gene transcription in mammalian cells.…”

A simplein vivofootprinting method to examine DNA-protein interactions over the yeastPYKUAS element

“…The RAP1 site (5'-CACCCAGACATC-3') is located in the PYK upstream activation sequence (UAS) at position -654 to -643 ( Figure 1; 22,23). Oligonucleotide primer P65 was used to examine the bottom strand for nucleotide base residues that were protected in vivo from DMS modification.…”