scholarly journals Mutant Caenorhabditis elegans RNA polymerase II with a 20,000-fold reduced sensitivity to alpha-amanitin.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 889-898
Author(s):  
T M Rogalski ◽  
M Golomb ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Cell Nuclei ◽  
Spliced Leader ◽  
Triton X ◽  
Alpha Amanitin

Abstract A doubly mutant ama-1(m118m526) gene results in an RNA polymerase (Rpo) II that is unusually resistant to alpha-amanitin. Rpo II activity in isolated Caenorhabditis elegans cell nuclei is inhibited 50% by alpha-amanitin at a concentration of 150 micrograms/ml, making this enzyme 150 times more resistant to the toxin than Rpo II from the singly mutant allele, ama-1(m118), 20,000 times more resistant than the wild-type Rpo II, and about six times more resistant to amanitin than is Rpo III. It was determined that the SL1 spliced leader precursor is transcribed by Rpo II, and this transcript was used to measure Rpo II activity. The Rpo II activity is unstable in vitro, and the mutant strain has a temperature-sensitive sterile phenotype. The highly resistant double mutant was selected among four million progeny of the mutagenized ama-1(m118) parent by its ability to grow and reproduce in 200 micrograms/ml amanitin in the presence of a permeabilizing agent, Triton X-100.

scholarly journals Lethal and amanitin-resistance mutations in the Caenorhabditis elegans ama-1 and ama-2 genes.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 409-422
Author(s):  
T M Rogalski ◽  
A M Bullerjahn ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Resistance Mutations ◽  
Recessive Lethal ◽  
New Gene ◽  
Alpha Amanitin

Abstract Mutants of Caenorhabditis elegans resistant to alpha-amanitin have been isolated at a frequency of about 1.6 x 10(-6) after EMS mutagenesis of the wild-type strain, N2. Four new dominant resistance mutations have been studied genetically. Three are alleles of a previously identified gene, ama-1 IV, encoding the largest subunit of RNA polymerase II. The fourth mutation defines a new gene, ama-2 V. Unlike the ama-1 alleles, the ama-2 mutation exhibits a recessive-lethal phenotype. Growth and reproduction of N2 was inhibited at a concentration of 10 micrograms/ml amanitin, whereas ama-2/+ animals were inhibited at 100 micrograms/ml, and 800 micrograms/ml was required to inhibit growth of ama-1/+ larvae. We have also determined that two reference strains used for genetic mapping, dpy-11(e224)V and sma-1(e30)V, are at least four-fold more sensitive to amanitin that the wild-type strain. Using an amanitin-resistant ama-1(m118) or ama-1(m322) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. The frequency of EMS-induced lethal ama-1 mutations is approximately 1.7 x 10(-3), 1000-fold higher than the frequency of amanitin-resistance alleles. Nine of the lethal alleles are apparent null mutations, and they exhibit L1-lethal phenotypes at both 20 degrees and 25 degrees. Six alleles result in partial loss of RNA polymerase II function as determined by their sterile phenotypes at 20 degrees. All but one of these latter mutations exhibit a more severe phenotype at 25 degrees C. We have also selected seven EMS-induced revertants of three different ama-1 lethals. These revertants restore dominant resistance to amanitin. The selection for revertants also produced eight new dominant amanitin resistance alleles on the balancer chromosome, nT1.

scholarly journals The Essential WD Repeat Protein Swd2 Has Dual Functions in RNA Polymerase II Transcription Termination and Lysine 4 Methylation of Histone H3

2004 ◽  
Vol 24 (7) ◽  
pp. 2932-2943 ◽  
Author(s):  
Hailing Cheng ◽  
Xiaoyuan He ◽  
Claire Moore
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Modification ◽  
Histone H3 ◽  
Temperature Sensitive ◽  
Repeat Protein ◽  
Tightly Coupled ◽  
Cleavage And Polyadenylation ◽  
Wd Repeat

ABSTRACT Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3′ end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum

1989 ◽  
Vol 9 (8) ◽  
pp. 3543-3547
Author(s):  
T W Nilsen ◽  
J Shambaugh ◽  
J Denker ◽  
G Chubb ◽  
C Faser ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Secondary Structure ◽  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Ascaris Lumbricoides ◽  
Parasitic Nematode ◽  
Secondary Structures ◽  
Spliced Leader ◽  
C Elegans

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

scholarly journals Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

1987 ◽  
Vol 7 (2) ◽  
pp. 586-594 ◽  
Author(s):  
M S Bartolomei ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Line ◽  
Resistance Mutation ◽  
Wild Type Mouse ◽  
Mutant Form ◽  
Wild Type ◽  
Gene Encoding ◽  
Base Pair Fragment ◽  
Alpha Amanitin

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-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 was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) 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 alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.

scholarly journals In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

1992 ◽  
Vol 12 (4) ◽  
pp. 1639-1651 ◽  
Author(s):  
S C Batson ◽  
R Sundseth ◽  
C V Heath ◽  
M Samuels ◽  
U Hansen
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Viral Dna ◽  
Dna Templates ◽  
Initiation Of Transcription ◽  
Transcription In Vitro ◽  
Alpha Amanitin

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals Ctr9, Rtf1, and Leo1 Are Components of the Paf1/RNA Polymerase II Complex

2002 ◽  
Vol 22 (7) ◽  
pp. 1971-1980 ◽  
Author(s):  
Cherie L. Mueller ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Double Mutant ◽  
Affinity Purification ◽  
Tata Binding Protein ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Pol Ii ◽  
Paf1 Complex ◽  
Transcriptional Start Sites

ABSTRACT The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Δ phenotypes. In particular, an rtf1Δ paf1Δ double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Δ single mutant. In addition, expression of the G1 cyclin CLN1, reduced nearly threefold in paf1Δ, is restored to wild-type levels in the rtf1Δ paf1Δ double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.

scholarly journals Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

2016 ◽  
Vol 90 (7) ◽  
pp. 3684-3693 ◽  
Author(s):  
Léa Meyer ◽  
Alix Sausset ◽  
Laura Sedano ◽  
Bruno Da Costa ◽  
Ronan Le Goffic ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type

ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

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