Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Santangelo, George M.; Tornow, Joanne; McLaughlin, Calvin S.; Moldave, Kivie

doi:10.1128/mcb.8.10.4217

Cited by 46 publications

(32 citation statements)

References 55 publications

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“…In yeast, transcription begins 40-100 nt downstream of a TATA box or equivalent promoter sequence (27). To minimize selection for transcription start sites, we placed the entire N18 sequence less than 40 nucleotides upstream of the HIS3 initiation codon.…”

Section: Resultsmentioning

confidence: 99%

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae

Zhou

Edelman

Mauro

2003

Proc. Natl. Acad. Sci. U.S.A.

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In previous studies, we demonstrated the sufficiency of short nucleotide sequences to facilitate internal initiation of translation in mammalian cells. By using a selection methodology, we have now identified comparable sequences in Saccharomyces cerevisiae. For these studies, a library of constructs expressing dicistronic mRNAs with the HIS3 gene as the second cistron and 18 random nucleotides in the intercistronic region was introduced into a yeast strain in which the endogenous HIS3 gene was deleted. Untransformed cells or those containing the parent construct failed to grow on medium lacking histidine. Intercistronic sequences recovered from cells that did grow were evaluated by using various criteria. Fifty-six of the 18-nt sequences (Ϸ1͞400,000) functioned as synthetic internal ribosome entry sites (IRESes). The 14 most active sequences allowed growth in the presence of 0.1-0.6 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product. In addition, eight sequences were identified that were not IRESes, but that enhanced HIS3 expression by an alternative mechanism that depended on the 5 end of the mRNA and appeared to involve either shunting or reinitiation. Comparisons among the 56 selected IRESes identified eight significant sequence matches containing up to 10 nucleotides. Many of the selected sequences also contained extensive complementary matches to yeast 18S rRNA, some at overlapping sites. The identification of cis sequences that facilitate translation initiation in yeast enables detailed biochemical and genetic analyses of underlying mechanisms and may have practical applications for bioengineering.

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Section: Resultsmentioning

confidence: 99%

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae

Zhou

Edelman

Mauro

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…2, there are two other rCNSs located in the EXP, rCNSs 3 and 4. Interestingly, rCNS 3 (composed of two adjacent sets of conserved peaks, rCNSs 3a and 3b) almost precisely corresponds to a previously identified bidirectional RNA polymerase II (pol II) promoter (36). No function has been attributed to this element, and it does not seem to promote transcription of any coding region.…”

Section: S Bayanusmentioning

confidence: 99%

Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting

Ganley

Hayashi

Horiuchi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Sequences involved in the regulation of genetic and genomic processes are primarily located in noncoding regions. Identifying such cis-acting sequences from sequence data is difficult because their patterns are not readily apparent, and, to date, identification has concentrated on regions associated with gene-coding functions. Here, we used phylogenetic footprinting to look for geneindependent noncoding elements in the ribosomal RNA gene repeats from several Saccharomyces species. Similarity plots of ribosomal intergenic spacer alignments from six closely related Saccharomyces species allowed the identification of previously characterized functional elements, such as the origin-of-replication and replication-fork barrier sites, demonstrating that this method is a powerful predictor of noncoding functional elements. Seventeen previously uncharacterized elements, showing high levels of conservation, were also discovered. The conservation of these elements suggests that they are functional, and we demonstrate the functionality of two classes of these elements, a putative bidirectional noncoding promoter and a series of conserved peaks with matches to the origin-of-replication core consensus. Our results paint a comprehensive picture of the functionality of the Saccharomyces ribosomal intergenic region and demonstrate that functional elements not involved in gene-coding function can be identified by using comparative genomics based on sequence conservation.gene amplification ͉ ribosomal RNA gene E ukaryote genomes contain large amounts of noncoding DNA, and it is clear that a diversity of functional elements are embedded in this noncoding DNA (1). However, finding such functional elements has been difficult because rules for their sequence patterns have not been established, and they are usually short and can have relaxed sequence requirements. One of the most powerful methods is locating areas of sequence conservation in intergenic regions between related organisms (2, 3), a technique known as phylogenetic footprinting (4). The rationale is that functional elements will show a greater level of selective constraint than the background sequence, thus standing out as ''footprints'' of high sequence conservation (5). To date, discovery of noncoding functional elements by phylogenetic footprinting has been restricted largely to those elements involved in controlling gene expression, such as promoters and enhancers. In this study, we used phylogenetic footprinting to look for noncoding functional elements that are not involved in the regulation of gene expression, and we call these elements gene-independent noncoding elements (NOCs). We looked for NOCs in the well studied ribosomal RNA gene repeats (rDNA; Fig. 1A) that contain both coding (rRNA) and noncoding [intergenic spacer (IGS)] regions. The rDNA has previously been shown to be involved in several gene-independent noncoding functions, such as origin-of-replication activity [ribosomal autonomous replicating sequence (rARS)] (6, 7), replication-fork barrier (RFB) act...

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“…Reporters used were pLG⌬AluXho (22) for CYC1 UAS1, which binds the activator HAP1; pLG265UP1 (18) for CYC1 UAS2, which binds the HAP2/3/4/5 heterotetrameric activator; HIS (1)66 (26) and HIS (2)14x2 (26), which bind GCN4; pCP0 (50), which contains the ADH1 promoter; and pdAT2 (36), which contains the thymidine-rich UAS of the DED1 gene. Other general yeast techniques were performed according to standard protocols (23).…”

Section: Methodsmentioning

confidence: 99%

ADA1, a Novel Component of the ADA/GCN5 Complex, Has Broader Effects than GCN5, ADA2, or ADA3

Horiuchi

Silverman

Piña

et al. 1997

Molecular and Cellular Biology

103

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The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here we describe the cloning, sequencing, and initial characterization of a novel ADA gene, ADA1. Similar to the previously isolated ada mutants, ada1 mutants display decreases in transcription from various reporters. Furthermore, ADA1 interacts with the other ADAs in the ADA/GCN5 complex as demonstrated by partial purification of the complex and immunoprecipitation experiments. We estimate that the complex has a molecular mass of approximately 2 MDa. Previously, it had been demonstrated that ada5 mutants displayed more severe phenotypic defects than the other ada mutants (G. A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996; S. M. Roberts and F. Winston, Mol. Cell. Biol. 16:3206-3213, 1996). ada1 mutants display defects similar to those of ada5 mutants and different from those of the other mutants with respect to promoters affected, inositol auxotrophy, and Spt ؊ phenotypes. Thus, the ADAs can be separated into two classes, suggesting that the ADA/GCN5 complex may have two separate functions. We present a speculative model on the possible roles of the ADA/GCN5 complex.RNA polymerase II (Pol II)-dependent transcription in eukaryotes requires general transcription factors and gene-specific transcriptional activators. The general factors include Pol II itself, the TATA binding protein (TBP), TFIIB, and other factors required for preinitiation complex formation at promoters. Activators bind to upstream activating sequences (UAS) or enhancers, which can be hundreds of base pairs upstream from the site of initiation of transcription, and stimulate transcription through their activation domains. In addition to these two types of factors, a third type whose members are referred to as mediators, coactivators, or adaptors has been isolated (3, 32, 44). These have been proposed to function by several mechanisms, including bridging interactions between activator proteins and basal factors and counteracting of the repressive effects on transcription by nucleosomes. Many factors believed to belong to this coactivator group form large heteromeric complexes. For example, the SWI/SNF complex is approximately 2 MDa in size (11,40,60) and functions by countering chromatin-mediated repression (14, 27). The suppressor of RNA polymerase B (SRB) complex is required for robust activated and basal transcription and interacts with basal factors to form an RNA Pol II holoenzyme complex (25,35,58). The TFIID complex consists of basal transcription factor TBP and associated TAF proteins which are thought to be required for activated transcription (15,51,59).Two other groups of factors which seem to function as adaptors or coactivators, the SPTs and the ADAs, were identified genetically in Saccharomyces cerevisiae. The SPT genes were isolated as suppressors of the auxotrophies resulting from transposon insertions at the promoters of various biosynthetic genes. The SPT genes fall into two classes based upon th...

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Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Cited by 46 publications

References 55 publications

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae

Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting

ADA1, a Novel Component of the ADA/GCN5 Complex, Has Broader Effects than GCN5, ADA2, or ADA3

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