Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

Guilfoyle, Tom J.

doi:10.1104/pp.58.4.453

Cited by 27 publications

(17 citation statements)

References 26 publications

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“…This is largely due to the fact that RNA polymerase II is the most abundant of all three RNA polymerases and is the most amenable to purification. Few studies have been done on the subunit structure of either RNA polymerases I or III from plants, although some preliminary subunit structures have been reported for RNA polymerase I in wheat (23), soybean (17), and cauliflower (15) and for RNA polymerase III in wheat (40). The results generally indicate subunit structures similar to those proposed for the cognate animal (31) and yeast (19) enzymes.…”

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confidence: 54%

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

Jendrisak¹

1981

Plant Physiol.

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A rapid and simple, large-scale method for the purification of DNAdependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH4)2SO4 precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000, 55,000, 38,000; 30,000-, 28,000; 25,000; 24,500, 20,500; 20,000; 19,500;, 17,800, and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.Studies with a variety of eukaryotic species indicate that transcription of the nuclear genome involves the participation of three classes of DNA-dependent RNA polymerases (EC 2.7.7.6) 5S rRNA and tRNAs (25).Eukaryotic nuclear RNA polymerases are multisubunit enzymes with aggregate mol wt of 500,000 to 750,000 (31). The subunit structures of RNA polymerases I, II, and III, as determined in animal systems (31) and yeast (19), are distinctly different. They superficially resemble each other in that they are all composed of two high molecular weight subunits (mol wt > 100,000), and several small subunits. Most of the subunits are different according to molecular weight. In yeast (19) and mammalian (31) systems there is evidence which indicates that a few low molecular weight subunits are shared in common by all three enzymes. In plant systems, only RNA polymerase II has been well-characterized according to subunit structure (27,28). This is largely due to the fact that RNA polymerase II is the most abundant of all three RNA polymerases and is the most amenable to purification. Few studies have been done on the subunit structure of either RNA polymerases I or III from plants, although some preliminary subunit structures have been reported for RNA polymerase I in wheat (23), soybean (17), and cauliflower (15) Here, methodology for the large-scale purification of RNA polymerase III from wheat germ is presented. This procedure has the advantage over other RNA polymerase purification scheme in that large quantities of starting material can be handled to yield mg quantities of highly purified enzyme. The subunit structure was analyzed by SDS-PAGE,2 and the results are compared with those reported by others (40). Significant discrepancies in the proposed subunit structure for wheat RNA polymerase III are discussed. In addition, evidence is presented for common subunits in all three RNA polymerases from wheat. MATERIALS AN...

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confidence: 54%

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

Jendrisak¹

1981

Plant Physiol.

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“…Promoterindependent (nonspecific) polymerase activity was measured using standard assays (31)(32)(33). Crude extract or column fraction (20 l) was mixed with 20 l of 2ϫ reaction mix containing 100 mM Hepes-KOH (pH 7.9), 100 mM KCl, 4 mM MnCl 2 , 1 mM each rATP, UTP, rCTP, 0.08 mM unlabeled rGTP, 2 mg͞ml BSA, 50 g͞ml sheared calf thymus DNA, 100 g͞ml ␣-amanitin (Sigma), and 10 Ci (1 Ci ϭ 37 GBq) ␣-labeled [ 32 P]rGTP ([ 32 P]UTP was used initially with similar results).…”

Section: Methodsmentioning

confidence: 99%

“…RNA pol III in Brassica is highly resistant to ␣-amanitin and is inhibited Ͻ50% even at 1 mg͞ml (33). Thus the contributions of pol I and pol III to the activity detected in DEAE fractions is not discriminated in the nonspecific assay.…”

Section: Development Of a Plant In Vitromentioning

confidence: 98%

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

Sáez‐Vasquez

Pikaard

1997

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

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RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inf lorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain Ϸ30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.Eukaryotic nuclear RNA polymerases interact with transcription factors to recognize gene promoters and initiate transcription. Many of the required factors can be purified individually and added back together to reconstitute transcription in vitro, allowing their identification and molecular characterization (1-7). However, proteins not required under a given set of assay conditions can be overlooked, though they may play important roles in vivo. The latter insight has come, in part, from the characterization of RNA pol II holoenzyme complexes in yeast and mammals (8)(9)(10)(11)(12). These complexes are several megadaltons in size and include pol II core enzyme subunits, most of the general transcription factors, and a growing list of additional activities including protein kinases (13), chromatin remodeling activities(14), and proteins involved in DNA repair (12).There has been at least one report of a pol I-containing fraction from rat cell-free extracts sufficient for accurate transcription in vitro (15), and several studies have shown that pol I transcription factors interact. For instance, the vertebrate pol I transcription factors UBF and SL1͞TIF-IB interact both in solution and on the DNA (16-18). Likewise, UBF and pol I cosediment in glycerol gradients and interact on Far-Western blots (19). Two additional factors, TIF-IA and TIF-IC copurify with pol I on multiple columns (20,21). Collectively, these results suggest that the pol I transcription factors might be associated with one another, possibly within a complex analogous to the pol II holoenzyme.Encouraged by brief reports that cell-free rRNA gene transcription could be achieved in plant extracts (22, 23), we set out to develop a cell-free system from broccoli (Brassica oleracea) to further our studies of rRNA gene regulation in Brassica and Arabidopsis (24-28). We show that a pol Icontaining activity sufficient for pr...

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“…Cauliflower inflorescence or curd is a dense mass of actively dividing cells that provides a rich source of transcriptionally active plant nuclei and RNA polymerases (Guilfoyle, 1976). In addition, the cauliflower inflorescence offers a source of plant material that is relatively free of chloroplasts and is commercially available.…”

Section: Purification Of Cauliflower R N a Polymerase I From Isolatedmentioning

confidence: 99%

Purification, subunit structure, and immunological properties of chromatin-bound ribonucleic acid polymerase I from cauliflower inflorescence

Guilfoyle¹

1980

Biochemistry

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Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

Cited by 27 publications

References 26 publications

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

Purification, subunit structure, and immunological properties of chromatin-bound ribonucleic acid polymerase I from cauliflower inflorescence

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