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. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of aL-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RP1215 gene.RNA polymerase II is a multisubunit enzyme that transcribes protein-coding genes in eucaryotes (see references 10, 34, and 45 for reviews). Although little is known about the functional role of individual subunits, the analysis of RNA polymerase II has been facilitated by use of the mushroom toxin at-amanitin. At low concentrations, uamanitin selectively inhibits RNA polymerase II from most species (32,'33, 35). a-Amanitin binds to RNA polymerase II with an equilibrium association constant of approximately 1010 M-1 and inhibits transcription by blocking elongation of the enzyme after the formation of a phosphodiester bond (13,54). The binding of a single toxin molecule to RNA polymerase II is sufficient to inhibit transcription (13).Mutations that give rise to an a-amanitin-resistant phenotype have been described for cell lines from several species (7,11,29,50,51). In these cell lines, the mutation conferring a-amanitin resistance renders the enzyme 5 to 1,000 times more resistant to a-amanitin. Hybrids of Chinese hamster ovary (CHO) cells that were constructed from a-amanitinsensitive and -resistant cells express both RNA polymerase II allelfs (36). However, when these hybrid lines are grown in the presence of a-amanitin, only the ao-amanitin-resistant enzyme is expressed (26). In this case, there appears to be enhanced degradation of the x-amanitin-sensitive protein together with a compensatory increase in the synthesis of the a-amanitin-resistant protein (27). Codominance of RNA polymerase II expression has also been observed in aamanititi-resistant L6 rat myoblast cells (17).a-Amanitin resistant Drosophila melanogaster (23) and Caenorhabditis elegans (46) strains have also been is...

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

“…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).…”

Specific HDV RNA-templated transcription by pol II in vitro

“…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...…”

“…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)+…”

Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

“…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).…”