The Predicted Presence of Large Helical Structural Variation in Yeast HIS4 Upstream Region is Correlated with General Amino Acid Control on the CYC1 Gene

Abstract

A series of CYC1 constructions in which the upstream promoter portion has been replaced by a variety of HIS4 synthetic fragments has demonstrated that the 5′ TGACTC 3′ repeat is crucial for conferring amino acid general control. Efficient regulation, however, is obtained only with fragments containing both the repeat and flanking sequences. Analysis of the flanks shows the presence of a 16 nucleotide long sequence composed of alternations of two purines and two pyrimidines between the upstream and downstream repeats. Such a sequence has very large twist angle variations. Homologous sequences are observed in HIS1, HIS3, and in TRP5 upstream regions between copies of the repeat. Sequences which confer special structural characteristics may aid in protein recognition of the promoter region.     

N6-methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA

The activity of eukaryotic promoters is highly sensitive to site-specific modifications by DNA methylations. We have used the E1A promoter of adenovirus type 12 (Ad12) DNA to investigate the effects of methylations at different promoter sites on its activity. The chloramphenicol acetyltransferase gene has served as an activity indicator. Activity of the E1A promoter is lost or markedly decreased by deoxycytidine methylation of two HpaII (5'-C-C-G-G-3') or seven HhaI (5'-G-C-G-C-3') sites upstream from the 3' located T-A-T-A signal. There are two T-A-T-A signals in the E1A promoter of adenovirus type 12 DNA, one T-A-T-T-A-T sequence starting at nucleotide 276 (5' located), a second T-A-T-T-T-A-A sequence starting at nucleotide 414 (3' located). Deoxycytidine methylations at two AluI (5'-A-G-C-T-3') sites downstream from the 5' located T-A-T-A signal have no effect on promoter activity. When one EcoRI (5'-G-A-A-T-T-C-3') or one TaqI (5'-T-C-G-A-3') sequence at 281 base-pairs upstream or 61 base-pairs downstream from the 5' located E1A T-A-T-A signal, respectively, is deoxyadenosine methylated, the promoter becomes inactive. Deoxyadenosine methylation at one MboI (5'-G-A-T-C-3') site, which is located 127 nucleotides downstream from the 5' located T-A-T-A signal, fails to decrease E1A promoter activity. There is no conspicuous anatomical relation of any of these sites to the two presumptive enhancer sequences in the E1A promoter. We conclude that 5-deoxymethylcytidine or N6-methyldeoxyadenosine residues have to be introduced at highly specific promoter sites to inactivate the promoter. These sites are probably different for different promoters.     

A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast

We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation. Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions. Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis. A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control. This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion. The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response. The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control.     

The repeated GC-rich motifs upstream from the TATA box are important elements of the SV40 early promoter.

We have constructed deletion and point mutations within the Simian virus 40 (SV40) early promoter region which contains two tandemly repeated 21 bp sequences and a related 22 bp sequence (the "upstream" 21 bp repeat region). After transfection into permissive CV-1 cells and non-permissive mouse 3T3-4E cells, the effect of the mutations on early gene expression was studied by measuring T-antigen production, using indirect immunofluoresence. Our results demonstrate that the 21 bp repeat region, and in particular the six GC-rich motifs 5'-CCGCCC-3' which are repeated in this region constitute an important element of the SV40 early promoter. Surprisingly, we found that the requirement for the 21 bp repeat region for early gene expression was partially fulfilled even when it was in the inverted orientation.