Blocking of the Initiation-to-Elongation Transition by a Transdominant RNA Polymerase Mutation

Kashlev, Mikhail; Lee, Jookyung; Zalenskaya, Katya; Nikiforov, Vadim; Goldfarb, Alex

doi:10.1126/science.1693014

Cited by 68 publications

(52 citation statements)

References 38 publications

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“…This phenotype is reminiscent of rpoB mutations in Escherichia coli which render the holoenzyme unable to clear a promoter and are dominant lethal when expressed in vivo (20-22, 32, 46). It is thought that this phenotype results from mutant polymerases occupying and occluding genes, thereby preventing their efficient transcription by wild-type polymerase (20,32,46).…”

Section: Discussionmentioning

confidence: 99%

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

Lennon

Wind

Saunders

et al. 1998

Molecular and Cellular Biology

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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 the rpb2-10 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.The elongation phase of transcription is an important control point for the regulation of gene expression (reviewed in references 4, 29, and 41). Several general elongation factors, including TFIIF, ELL, and elongin (SIII), are able to increase the overall rate of transcription elongation of RNA polymerase II (PolII) in vitro (7,13,26,37). SII enables PolII to transcribe through a variety of transcriptional blockages, including intrinsic arrest sites and nucleoprotein complexes (reviewed in references 29 and 41). SII reactivates arrested PolII complexes by binding to the enzyme and activating a nascent RNA cleavage activity, which eventually results in polymerase escape (reviewed in reference 28). It has been hypothesized that elongation factors such as TFIIF reduce the frequency of arrest by reducing the dwell time of PolII at arrest sites (6,15).Little is known about gene sequences that block transcription and the interaction of general elongation factors with PolII complexes in vivo. In yeast, mutation or disruption of the gene encoding SII, DST1 (gene names in this study are those designated in the Saccharomyces Genome Database; DST1 is also known as PPR2), renders cells sensitive to the base analog 6-azauracil (6AU) (19,23,24). 6AU depletes cellular levels of the RNA precursors UTP and GTP (12). Supplementing the drug-containing medium with uracil or guanine reverses this phenotype, suggesting that the drug inhibits growth because of the reduction in nucleoside triphosphate levels and thereby impairs transcription elongation (3, 12). The sensitivity of yeast to 6AU after disruption of DST1 may indicate an increased requirement by RNA polymerase for elongat...

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Section: Discussionmentioning

confidence: 99%

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

Lennon

Wind

Saunders

et al. 1998

Molecular and Cellular Biology

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“…The catalytic core of the bacterial enzyme, capable of normal elongation and termination, is made of two large ␤ and ␤Ј subunits and a dimer of ␣ subunits, which correspond to RPB2, RPB1, and a heterodimer of two RPB3/RPB5 subunits in the yeast Pol II, respectively (23,36). There is a vast pool of already known mutants of E. coli RNAP with altered elongation properties, and a well established genetic system makes it easy to obtain and characterize novel mutants (51). Moreover, the biochemical system allows for reconstitution of E. coli RNAP from either separately purified subunits or even separate domains within the subunits, which extends the list of manipulations possible with mutant RNAPs in vitro (52).…”

Section: Nucleosomes Form Similar Barriers To Assembled and Promoter-mentioning

confidence: 99%

Bacterial Polymerase and Yeast Polymerase II Use Similar Mechanisms for Transcription through Nucleosomes

Walter

Kireeva

Studitsky

et al. 2003

Journal of Biological Chemistry

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We have previously shown that nucleosomes act as a strong barrier to yeast RNA polymerase II (Pol II) in vitro and that transcription through the nucleosome results in the loss of an H2A/H2B dimer. Here, we demonstrate that Escherichia coli RNA polymerase (RNAP), which never encounters chromatin in vivo, behaves similarly to Pol II in all aspects of transcription through the nucleosome in vitro. The nucleosome-specific pausing pattern of RNAP is comparable with that of Pol II. At physiological ionic strength or lower, the nucleosome blocks RNAP progression along the template, but this barrier can be relieved at higher ionic strength. Transcription through the nucleosome by RNAP results in the loss of an H2A/H2B dimer, and the histones that remain in the hexasome retain their original positions on the DNA. The results were similar for elongation complexes that were assembled from components (oligonucleotides and RNAP) and elongation complexes obtained by initiation from the promoter. The data suggest that eukaryotic Pol II and E. coli RNAP utilize very similar mechanisms for transcription through the nucleosome. Thus, bacterial RNAP can be used as a suitable model system to study general aspects of chromatin transcription by Pol II. Furthermore, the data argue that the general elongation properties of polymerases may determine the mechanism used for transcription through the nucleosome.In eukaryotes, the DNA within the nucleus is packaged by histones into nucleosomes. In each nucleosome core, 146 bp of DNA are wrapped in about 1.7 superhelical coils around an octamer containing two molecules each of histones H2A, H2B, H3, and H4. The nucleosome core has a tripartite structure, with the H3/H4 tetramer organizing the central ϳ90 bp of nucleosomal DNA and two H2A/H2B dimers bound to the ends of the nucleosomal DNA (1).Nucleosomes are present in vivo even when genes are actively transcribed by RNA polymerase II (Pol II), 1 indicating that even if nucleosomal structure is disrupted during transcription, recovery occurs almost immediately after passage of the enzyme (see Refs. 2 and 3 for reviews). Analysis of a Pol II encounter with a mononuclesome resulted in the discovery that the nucleosome acts as a potent barrier to the core Pol II enzyme in vitro, which cannot be relieved even when transcription is conducted in the presence of general elongation factors (4, 5). More recently, it has been shown that transcription through the nucleosome by Pol II results in the loss of an H2A/H2B dimer without changing the position of the histones on the DNA (6). The bacteriophage SP6 and T7 RNA polymerases have been extensively used as model systems for the in vitro analysis of transcription through the nucleosome (7). The properties of nucleosomal transcription by the multisubunit Pol II and the single-subunit phage SP6 RNAP are dramatically different. The nucleosome is a relatively weak barrier to transcription by bacteriophage RNAP (8 -10), and during transcription, the complete histone octamer is transferred from in front of ...

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“…although titred antibodies were used in the immunoprecipitation. The fact that the B3-4 segment is accessible is in keeping with the catalytic role of this region in the enzymatic reaction (Grachev et al 1987(Grachev et al , 1989Kashlev et al 1990;Lee et al 1991). Taken together, we would suggest the N-terminal half of ␤, in particular the region specified by segment B2, is generally 'buried' within the enzyme complex (previous studies have shown that a small number of particular epitopes are accessible; see, for instance, Severinov et al 1993).…”

Section: Figurementioning

confidence: 67%

Function in vivo of separate segments of the β subunit of Escherichia coli RNA polymerase

Trigwell

Glass

1998

Genes to Cells

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Background: Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed ␤ and ␤ 0 , are conserved throughout nature. The ␤ subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the ␤ subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the ␤ subunit of RNA polymerase. To this end, the ␤ structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half ' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed

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Blocking of the Initiation-to-Elongation Transition by a Transdominant RNA Polymerase Mutation

Cited by 68 publications

References 38 publications

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

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

Bacterial Polymerase and Yeast Polymerase II Use Similar Mechanisms for Transcription through Nucleosomes

Function in vivo of separate segments of the β subunit of Escherichia coli RNA polymerase

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