Chapter 15 Protein—DNA Interactions in Vivo—Examining Genes in Saccharomyces cerevisiae and Drosophila melanogaster by Chromatin Footprinting

Hull, Melissa W.; Thomas, Graham H.; Huibregtse, Jon M.; Engelke, David R.

doi:10.1016/s0091-679x(08)60581-6

Cited by 17 publications

(15 citation statements)

References 40 publications

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“…After electrophoresis, the gels were dried and all four species of DNA (bound and free test DNA, bound and free reference Analysis of chromatin structure. The chromatin structure of the MET16 promoter was analyzed essentially as described by Hull et al (19). Cultures were prepared exactly as described for the Northern blot experiments (see above) and harvested at a density of approximately 10 7 cells per ml (2 to 3 h after the nutritional shift).…”

Section: Methodsmentioning

confidence: 99%

“…Digestion was allowed to proceed for 3 min and then terminated with an equal volume of stop solution (1 M NaCl, 50 mM Tris-Cl [pH 7.9], 2% sodium dodecyl sulfate, 50 mM EDTA). Genomic DNA was isolated as described previously (19), digested to completion with EcoRI and XbaI, and separated by agarose gel electrophoresis. The DNA was transferred to a nylon membrane and analyzed by indirect end labeling (42), with the same EcoRI-ClaI MET16 probe as was used for Northern analysis.…”

Section: Methodsmentioning

confidence: 99%

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Role of the Saccharomyces cerevisiae General Regulatory Factor CP1 in Methionine Biosynthetic Gene Transcription

O’Connell

Surdin-Kerjan

Baker

1995

Molecular and Cellular Biology

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Saccharomyces cerevisiae general regulatory factor CP1 (encoded by the gene CEP1) is required for optimal chromosome segregation and methionine prototrophy. MET16-CYC1-lacZ reporter constructs were used to show that MET16 5-flanking DNA contains a CP1-dependent upstream activation sequence (UAS). Activity of the UAS required an intact CP1-binding site, and the effects of cis-acting mutations on CP1 binding and UAS activity correlated. In most respects, MET16-CYC1-lacZ reporter gene expression mirrored that of chromosomal MET16; however, the endogenous gene was found to be activated in response to amino acid starvation (general control). The latter mechanism was both GCN4 and CP1 dependent. MET25 was also found to be activated by GCN4, albeit weakly. More importantly, MET25 transcription was strongly CP1 dependent in gcn4 backgrounds. The modulation of MET gene expression by GCN4 can explain discrepancies in the literature regarding CP1 dependence of MET gene transcription. Lastly, micrococcal nuclease digestion and indirect end labeling were used to analyze the chromatin structure of the MET16 locus in wild-type and cep1 cells. The results indicated that CP1 plays no major role in configuring chromatin structure in this region, although localized CP1-specific differences in nuclease sensitivity were detected.General regulatory factors form a family of sequence-specific DNA-binding proteins of Saccharomyces cerevisiae. Although structurally dissimilar, all of these factors are moderately abundant proteins with many genomic sites of interaction and are involved in diverse genetic processes. Members of the family include CP1, RAP1, REB1, and ABF1; with the exception of CP1, all are essential proteins. General regulatory factors have been shown to be associated with chromosome origins of replication (ABF1), telomeres (RAP1), and centromeres (CP1). In addition to their association with loci involved in chromosome maintenance, all have been implicated to some degree as positive and negative regulators of transcription (6,7,9). CP1 was the first member of the general regulatory factor family to be identified (5) and was originally characterized as an abundant protein, present in yeast extracts, that bound a conserved element of yeast centromeres referred to as centromere DNA element I (CDEI). Cloning and sequencing of CEP1, the gene encoding CP1, revealed the presence of a basic region-helix-loop-helix domain, a DNA-binding and dimerization motif common to a family of proteins involved in transcriptional regulation (8,26). The analysis of mutants in which the CEP1 gene had been disrupted confirmed the proposed role of CP1 in chromosome segregation and provided the first evidence that it was involved in other processes as well. Strains lacking a functional CEP1 gene product were found to display an array of phenotypes including an increase in the frequency of mitotic and meiotic chromosome missegregation, slow growth, and a requirement for methionine (3,8,23,26).The physiological cause of cep1 methionine auxotrophy is e...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Role of the Saccharomyces cerevisiae General Regulatory Factor CP1 in Methionine Biosynthetic Gene Transcription

O’Connell

Surdin-Kerjan

Baker

1995

Molecular and Cellular Biology

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“…Primer extension for 50 min at 42°C was performed using oligonucleotides labeled with ½g-32 PATP (6000 Ci/mmol) using PNK (NEB) (Supplemental Table S2). Dideoxy sequencing ladders were generated using Rpr1 DNA from a pUC19 plasmid (Hull et al 1991). After this, EtOH precipitation samples were resuspended in 23 FEXBS and separated on a 6% denaturing polyacrylamide gel for visualization.…”

Section: Cross-linkingmentioning

confidence: 99%

Binding and cleavage of unstructured RNA by nuclear RNase P

Marvin

Walker

Fierke³

et al. 2011

RNA

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Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 59 leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pretRNA Tyr , poly(U) 50 RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in ''Conserved Region I,'' although the exact positions vary. Additional contacts between poly(U) 50 and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.

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“…This procedure probed in vivo assembled chromatin by lysing yeast cells directly into a solution of DNase I. For comparison, purified genomic DNA was digested with DNase I. Cleavage sites were detected by primer extension (27). No obvious DNase I footprint was observed between the SNR14 TATA box and start site (Fig.…”

Section: Conserved Sequence Elementsmentioning

confidence: 99%

Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model

Kuehner

Brow

2006

Journal of Biological Chemistry

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Initiation of transcription by RNA polymerase II (RNAP II) onSaccharomyces cerevisiae messenger RNA (mRNA) genes typically occurs at multiple sites 40 -120 bp downstream of the TATA box. The mechanism that accommodates this extended and variable promoter architecture is unknown, but one model suggests that RNAP II forms an open promoter complex near the TATA box and then scans the template DNA strand for start sites. Unlike most proteincoding genes, small nuclear RNA gene transcription starts predominantly at a single position. We identify a highly efficient initiator element as the primary start site determinant for the yeast U4 small nuclear RNA gene, SNR14. Consistent with the scanning model, transcription of an SNR14 allele with tandemly duplicated start sites initiates primarily from the upstream site, yet the downstream site is recognized with equivalent efficiency by the diminished population of RNAP II molecules that encounter it. A quantitative in vivo assay revealed that SNR14 initiator efficiency is nearly perfect (ϳ90%), which explains the precision of U4 RNA 5 end formation. Initiator efficiency was reduced by cis-acting mutations at ؊8, ؊7, ؊1, and ؉1 and trans-acting substitutions in the TFIIB B-finger. These results expand our understanding of RNAP II initiation preferences and provide new support for the scanning model. Eukaryotes rely on RNA polymerase II (RNAP II)2 to synthesize all messenger RNAs (mRNAs) and most of the small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) encoded within their nuclear genomes. Efficient and accurate transcription initiation is vital to ensure the proper expression and function of these RNAs. The recruitment of RNAP II to gene promoters is mediated through the assembly of a pre-initiation complex (PIC). RNAP II accessory proteins provide promoter specificity and the structural core for assembly of the PIC. These accessory proteins include the general transcription factors TFIID, TFIIB, TFIIF, TFIIE, and TFIIH (1-3). Some transcription factors engage in sequence-specific contacts with core promoter elements (4); one of the most fundamental interactions for PIC assembly is between the TATA-binding protein subunit of TFIID and the TATA box (5-6). In a stepwise model for PIC assembly, TATA-binding protein binding is followed by the addition of TFIIB, RNAP II-TFIIF, TFIIE, and TFIIH (7).In metazoans, the assembly of a PIC at the TATA box results in start site selection 25-30 bp downstream (4). The architecture of the PIC is such that the transcription start site is placed precisely within the active center of RNAP II (8 -9). In the yeast Saccharomyces cerevisiae, RNAP II initiation typically occurs at multiple sites at variable distances from the TATA box, with most start sites ranging from 40 to 120 bp downstream of the TATA box (10). The initiation mechanism that accommodates this extended and variable promoter architecture is unknown, but it does not appear to be dependent on assembling the yeast PIC in a manner different from that of metazoans. Yeast p...

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Chapter 15 Protein—DNA Interactions in Vivo—Examining Genes in Saccharomyces cerevisiae and Drosophila melanogaster by Chromatin Footprinting

Cited by 17 publications

References 40 publications

Role of the Saccharomyces cerevisiae General Regulatory Factor CP1 in Methionine Biosynthetic Gene Transcription

Role of the Saccharomyces cerevisiae General Regulatory Factor CP1 in Methionine Biosynthetic Gene Transcription

Binding and cleavage of unstructured RNA by nuclear RNase P

Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model

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