1987
DOI: 10.1128/mcb.7.2.586
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Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

Abstract: RNA polymerase II is inhibited by the mushroom toxin a-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to ax-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form wa,s 500 times more resistant to aL-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RP1I215) gene (J. A. Ahearn, M. S. Bartolomei, M. L. West, and J. L. Co… Show more

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Cited by 98 publications
(56 citation statements)
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“…Kawagishi et al 1993;Azuma et al 1993) and thus are thought to form an enzyme core with the catalytic function of RNA synthesis. Supporting this prediction are the findings that the target of -amanitin, the potent inhibitor of RNA synthesis by eukaryotic RNA polymerase II, is located on conserved region F in subunit 1 of eukaryotic RNA polymerase II (Bartolomei & Corden 1987;Rogalski et al 1990;Chen et al 1993); the substrate-binding site is located in region I conserved between S. cerevisiae RPB2 and E. coli subunit (Treich et al 1992); and S. cerevisiae RPB3 appears to be involved in an initial step of the subunit assembly of RNA polymerase II (Kolodziej & Young 1991) as in the case of prokaryotic RNA polymerase (Ishihama 1981).…”
Section: Discussionmentioning
confidence: 54%
“…Kawagishi et al 1993;Azuma et al 1993) and thus are thought to form an enzyme core with the catalytic function of RNA synthesis. Supporting this prediction are the findings that the target of -amanitin, the potent inhibitor of RNA synthesis by eukaryotic RNA polymerase II, is located on conserved region F in subunit 1 of eukaryotic RNA polymerase II (Bartolomei & Corden 1987;Rogalski et al 1990;Chen et al 1993); the substrate-binding site is located in region I conserved between S. cerevisiae RPB2 and E. coli subunit (Treich et al 1992); and S. cerevisiae RPB3 appears to be involved in an initial step of the subunit assembly of RNA polymerase II (Kolodziej & Young 1991) as in the case of prokaryotic RNA polymerase (Ishihama 1981).…”
Section: Discussionmentioning
confidence: 54%
“…Antigenomic (AG) HDV RNA serves as a template for RNA synthesis by pol II+ A: Schematic representation of the full-length singlestranded circular AG HDV RNA folded into an unbranched rod-like structure+ The AG ribozyme domain (rectangle) and its site of cleavage (circle) are indicated+ The arrow depicts the open reading frame for HDAg+ B: AG200, the segment of AG HDV RNA used as a template in NE transcription reactions+ C: RNA synthesis in wt (lanes 1-6) and PMG (lanes 7-12) HeLa NE usingAd2MLP DNA (lanes 1-3 and 7-9) or HDV RNA (lanes 4-6 and 10-12) as templates+ Transcription reactions monitored by incorporation of a-32 P-GTP into newly synthesized RNAs were carried out under standard conditions (lanes 1, 4, 7, and 10), in the presence of 1 mg/mL a-amanitin (lanes 2, 5, 8, and 11), or 20 mg/mL actinomycin D (lanes 3, 6, 9, and 12)+ Products were resolved in a 5% (Wang et al+, 1986), flanked at its 59 end by a 59-nt polylinker sequence+ Synthesis of ;400-nt-long product is only modestly inhibited in the presence of actinomycin D (Fig+ 1C, lane 6), consistent with the RNA-, rather than DNA-templated process, but is strongly inhibited by a-amanitin (Fig+ 1C, lane 5), suggesting pol II involvement+ In contrast, template labeling is not significantly affected by either of these toxins (Fig+ 1C, lanes 5 and 6)+ To confirm pol II involvement in the RNA-templated transcription reaction, NE was prepared from PMG HeLa cell line that contains a copy of the mouse RPII215 gene encoding an a-amanitin resistant variant of the largest subunit of pol II (Bartolomei & Corden, 1987; J+ Corden, pers+ comm+)+ In control experiments using the Ad2MLP DNA template, transcription in PMG NE was reduced approximately threefold in the presence of a-amanitin (Fig+ 1C, lanes 7 and 8)+ This result was expected, because in addition to the integrated mutant gene, PMG cells still contain the endogenous a-amanitin-sensitive pol II alleles+ RNA-templated transcription in the PMG NE was also reduced two-to threefold (Fig+ 1C, lanes 10 and 11), as in the case of the DNA-templated reaction (Fig+ 1C, lanes 7 and 8)+ These results strongly suggest that pol II in the HeLa NE can use HDV RNA as a template+ However, additional NE components must also be required, since a purified pol II preparation (gift of A+ Hoffmann and R+G+ Roeder) did not support RNA transcription under these conditions (data not shown)+ Further studies will be required to determine if any of the known protein components of pol II machinery are involved in this RNA-templated process+ RNA synthesis using all these templates is sensitive to a-amanitin, consistent with the involvement of pol II in these reactions+ The apparent sizes of generated RNA products provided an initial information concerning the start site of transcription+ As expected, templates with deletions in the 59 half of the molecule, for example, AG130 and AG138 (Fig+ 2B, lanes 2 and 5, respectively), yielded products that migrated faster than that of AG128 RNA, and the change in product mobility correlated well with the size of the introduced deletion (;30 nt)+ However, deletions in the 39 portion of the template did not affect gel mobility of the corresponding products (compare AG128 with AG131 and AG132, Fig+ 2B, lanes 1, 3, and 4)+ These results suggest that the start site of the observed transcription is located upstream (59) of the region deleted in AG132 RNA and downstream (39) of the region deleted in AG138 RNA, that is, near the loop of the hairpin template+ Consistent with this notion, double deletions in both 59 and 39 regions of the template (e+g+, AG129 and AG103, Fig+ 2B, lanes 6 and 7) affect the mobility of products to the same extent as single deletions in the 59 region only (e+g+, AG130 and AG138, Fig+ 2B, lanes 2 and 5)+ Further deletions of the 218-nt AG103 RNA from either the 59 or 39 end inactivated these templates (data not shown), and thus AG103 RNA was used as a s...…”
Section: Pol II In Hela Cell Ne Utilizes Segments Of Ag Hdv Rna As Tementioning
confidence: 98%
“…Here we describe an in vitro system based on HeLa cell nuclear extracts (NE), in which a specific segment of AG HDV RNA serves as a template for efficient and highly specific RNA synthesis+ This RNA-dependent RNA synthesis involves pol II, as indicated by a-amanitin sensitivity of the process+ The pol II involvement is further documented by partial a-amanitin resistance of the RNA synthesis in PMG cell extracts that contain an a-amanitin resistant allele of the largest subunit of pol II (Bartolomei & Corden, 1987; J+L+ Corden, pers+ comm+)+ The RNA product generated in these reactions represents a chimeric molecule composed of the newly transcribed RNA covalently attached to the 59 portion of the template+ Selection of the start site for transcription is remarkably specific and determined by the secondary structure of the RNA template, rather than by its primary sequence+ This mode of RNA synthesis is reminiscent of the RNA cleavage-extension reactions observed in vitro for RNA polymerase-arrested complexes (reviewed by Uptain et al+, 1997)+…”
Section: Introductionmentioning
confidence: 99%
“…This fungal toxin blocks TFIIS-stimulated transcript cleavage, without significantly affecting the rate of intrinsic cleavage. The toxin, ␣-amanitin, binds tightly to the largest subunit of RNA polymerase II (47,69) and dramatically slows elongation and pyrophosphorolysis (48,49). However, it appears to interfere with translocation rather than phosphodiester bond formation (70).…”
Section: Discussionmentioning
confidence: 99%