Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

Kasher, M S; Pintel, David J.; Ward, David C.

doi:10.1128/mcb.6.9.3117

Cited by 35 publications

(16 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The yield of fixation was about 30% as determined by the amount of radioactivity retained on the column and was not improved by increasing the contact times. The reason for this low efficiency is unclear, but it should be pointed out that similar results were obtained by Kasher et al [9] with avidinagarose and biotin-1 1 -dUTP. Moreover, essentially quantitative fixation can be achieved provided that several biotin-1 1-dUTP molecules are attached to the DNA fragment, using terminal deoxynucleotide transferase (Jacques Htrisst, personal communication).…”

Section: Resultssupporting

confidence: 65%

See 1 more Smart Citation

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

1987

View full text Add to dashboard Cite

We describe a method for the purification of proteins binding to specific DNA sites based on the strong interaction between streptavidin and biotin. We tested the efficiency of this method using the Escherichia coli lactose operon operator-repressor system. dUTP coupled to biotin is incorporated into a DNA fragment containing the lactose operator. A crude E. coli extract is first incubated with the biotinylated fragment and the reaction mixture is filtered on a streptavidin-agarose column. Proteins retained on the column are either eluted alone by high salt or isopropyl p-D-thiogalactoside, or as a complex with the DNA site by enzymatic digestion of the DNA. We thus obtained a 3400-fold enrichment of the repressor complexed to the operator in one step. The method is simple and makes use of commercially available reagents. The large concentration of biotin-binding sites of the streptavidin-agarose matrix (0.1 pmol/ml packed gel) provides a very high capacity for the concentration and purification of large amounts of proteins. The advantage of this method for the detection and purification of other DNA-binding proteins is discussed.Gene transcription is regulated by both cis and transacting factors. Regulatory sequences located either near the transcription start site, such as promoters, or variably located such as enhancers, have been identified. Cell-type-specific transcription is presumably conferred by the interaction of selective proteins with certain of these sequences. Genetic methods, such as cell fusion or transfection experiments, provide an indirect approach in the study of trans-acting factors, while in vitro transcription assays [l], DNA protection 121, gel retardation [3, 41 and Western blotting [5] methods have yielded direct information on the interaction between sitespecific binding proteins and DNA regulatory sequences. A complete characterisation of these factors, however, necessarily depends on their purification. Since they are generally present at very low cellular concentration, powerful methods such as affinity chromatography using the target DNA must be employed.Several authors have recently reported different methods to obtain a DNA site matrix. Rosenfeld and Kelly [6] coupled a plasmid bearing multiple recognition sites to cellulose. Alternatively, Kadonaga and Tjian [7] used a polymerised synthetic oligonucleotide. Levens and Howley [8] fixed a / Igalactosidase -lac-repressor fusion protein to anti-(pga1actosidase)-Sepharose. The DNA sequence of interest was ligated to the lactose operator sequence and thus retained on the column. Kasher et al.[9] used biotin-cellulose and a biotinylated DNA fragment to obtain a DNA matrix via streptavidin bridges.To various degrees all these methods suffer from difficulties due to competition between non-specific DNAbinding proteins and the protein of interest for binding to the target DNA, and to inactivation of the purified protein. We present here a novel method designed to minimize these problems, uniting individual advantages of the preceding met...

show abstract

Section: Resultssupporting

confidence: 65%

“…The DNA sequence of interest was ligated to the lactose operator sequence and thus retained on the column. Kasher et al [9] used biotin-cellulose and a biotinylated DNA fragment to obtain a DNA matrix via streptavidin bridges.…”

mentioning

confidence: 99%

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

1987

View full text Add to dashboard Cite

show abstract

“…To this end, we developed an immobilized-ribosomal DNA (rDNA) promoter tem-plate assay similar to those which previously have proven instrumental in the analysis of Pol III and Pol II PICs (2,26,34,39). This assay allowed us to capture and purify Pol I PICs from nuclear extracts and study their fate in the transcription cycle.…”

mentioning

confidence: 99%

A Step Subsequent to Preinitiation Complex Assembly at the Ribosomal RNA Gene Promoter Is Rate Limiting for Human RNA Polymerase I-Dependent Transcription

Panov

Friedrich

Zomerdijk

2001

Molecular and Cellular Biology

View full text Add to dashboard Cite

The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contained the factors required to support initiation of transcription, SL1, upstream binding factor (UBF), and Pol I. We have demonstrated that, throughout a single round of transcription, SL1 and UBF remain promoter bound. Moreover, the promoterbound SL1 and UBF retain the ability to function in transcription initiation. SL1 has a central role in the stable association of the PIC with the promoter DNA. The polymerase component of the PIC is released from the promoter during transcription yet is efficiently recycled and able to reinitiate from "poised" promoters carrying SL1 and UBF, since the PICs captured on the immobilized templates sustained multiple rounds of transcription. Kinetic analyses of initiation of transcription by Pol I revealed that Pol I-dependent transcription is rate limited in a step subsequent to recruitment and assembly of Pol I PICs. The rate of RNA synthesis is primarily determined by the rates at which the polymerase initiates transcription and escapes the promoter, referred to as promoter clearance. This rate-limiting step in Pol I transcription is likely to be a major target in the regulation of rRNA gene expression.

show abstract

“…These include the well-characterized RNA polymerase III and two subcellular fractions which have been functionally designated TFIIIB and TFIIIC. A component in TFIIIC has been shown by physical methods to interact directly with DNA sequences around the B-block consensus element (8,15,22 stoichiometric interaction of TFIIIB with the TFIIIC-VA gene complex, followed by rapid association of RNA polymerase III to form a complete preinitiation complex (8,20).…”

mentioning

confidence: 99%

“…Studies with chromatographically purified transcription complexes have shown that there is a * Corresponding author. stoichiometric interaction of TFIIIB with the TFIIIC-VA gene complex, followed by rapid association of RNA polymerase III to form a complete preinitiation complex (8,20).…”

mentioning

confidence: 99%

Multiple proteins bind to VA RNA genes of adenovirus type 2.

Dyke¹,

Roeder²

1987

Mol. Cell. Biol.

View full text Add to dashboard Cite

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA-Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200 (3,13,18). Spacing between the A and B blocks is important for maintaining transcriptional activity (3, 7). At least three cellular proteins are necessary to reconstitute transcription from the VA genes in vitro (37). These include the well-characterized RNA polymerase III and two subcellular fractions which have been functionally designated TFIIIB and TFIIIC. A component in TFIIIC has been shown by physical methods to interact directly with DNA sequences around the B-block consensus element (8,15,22 stoichiometric interaction of TFIIIB with the TFIIIC-VA gene complex, followed by rapid association of RNA polymerase III to form a complete preinitiation complex (8,20).During the course of adenovirus infection, the relative rates of synthesis of the two VA RNAs differ markedly (38). Transcription from both genes commences shortly after infection, with both RNAs being produced at roughly equivalent rates. This pattern continues until the onset of viral DNA replication, approximately 8 to 10 h postinfection, at which time VAII synthesis levels off while VAI synthesis accelerates. Thus, at late times the ratio of VAI to VAII RNA present may be as high as 40:1 (40). The transcriptional control underlying the differential expression of the two VA genes is not fully understood. Evidence points toward competition between the two VA promoters for a transcription factor present in limiting quantities (4).To understand the mechanistic basis for differential regulation of the VA genes, as well as the roles of sequence and factors in the mechanism of RNA polymerase ITT-mediated specific transcription, we undertook a study of those proteins which interact with the VA genes and their effects on transcription. With fractionated HeLa cell nuclear extracts and both DNase I (16) and methidiumpropyl-EDTA-Fe(II) (MPE) (35, 41) cleavage protection methodologies, three proteins were shown to interact specifically, yet differentially, with the two VA genes. Two of these proteins were identified as the polymerase III transcription factor TFIIIC and the DNA replication protein nuclear factor I (NFI), whereas the third, a VA gene binding protein designated VBP, was previously unrecognized. The possible functions of these proteins in VA transcription were analyzed in reconstituted systems. Whereas NFI exhibited no...

show abstract

Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

Cited by 35 publications

References 28 publications

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

A Step Subsequent to Preinitiation Complex Assembly at the Ribosomal RNA Gene Promoter Is Rate Limiting for Human RNA Polymerase I-Dependent Transcription

Multiple proteins bind to VA RNA genes of adenovirus type 2.

Contact Info

Product

Resources

About