Mammalian RNA Polymerase Research Articles

Transcriptional Inhibition by an Oxidized Abasic Site in DNA

2-Deoxyribonolactone (dL) is an oxidized abasic site in DNA that can be induced by gamma-radiolysis, ultraviolet irradiation, and numerous antitumor drugs. Although this lesion is incised by AP endonucleases, suggesting a base-excision repair mechanism for dL removal, subsequent excision and repair synthesis by DNA polymerase beta is inhibited due to accumulation of a protein-DNA cross-link. This raises the possibility that additional repair pathways might be required to eliminate dL from the genome. Transcription-coupled repair (TCR) is a pathway of excision repair specific to DNA lesions present in transcribed strands of expressed genes. A current model proposes that transcription arrest at the site of DNA damage is required to initiate TCR. In support of this model, a strong correlation between transcription arrest by a lesion in vitro and TCR of the lesion in vivo has been found in most cases analyzed. To assess whether dL might be subject to TCR, we have studied the behavior of bacteriophage T3 and T7 RNA polymerases (T3RNAP, T7RNAP) and of mammalian RNA polymerase II (RNAPII) when they encounter a dL lesion or its "caged" precursor located either in the transcribed or in the nontranscribed strand of template DNA. DNA plasmids containing a specifically located dL downstream of the T3, T7 promoter or the Adenovirus major late promoter were constructed and used for in vitro transcription with purified proteins. We found that both dL and its caged precursor located in the transcribed strand represented a complete block to transcription by T3- and T7RNAP. Similarly, they caused more than 90% arrest when transcription was carried out with mammalian RNAPII. Furthermore, RNAPII complexes arrested at dL were subject to the transcript cleavage reaction mediated by elongation factor TFIIS, indicating that these complexes were stable. A dL in the nontranscribed strand did not block either polymerase.

The Mammalian RNA Polymerase II C-Terminal Domain Interacts with RNA to Suppress Transcription-Coupled 3′ End Formation

RNA polymerase II plays a critical role not only in transcription of mRNA precursors but also in their subsequent processing. This later function is mediated primarily by the C-terminal domain (CTD) of the enzyme's largest subunit, a unique, repetitive structure conserved throughout eukaryotes and known to interact with a number of different proteins during the transcription cycle. Here, we show that the mammalian CTD also interacts with RNA in a sequence-specific manner. We use a variety of RNA binding assays, including SELEX, to characterize the interaction in vitro and a modified chromatin immunoprecipitation (ChIP) assay to provide evidence that it also occurs in vivo. Transfection assays with the CTD binding consensus situated downstream of a polyadenylation signal indicate that the sequence can suppress mRNA 3' end formation and transcription termination, and in vitro assays indicate that the inhibition of processing is CTD dependent. Our results provide an unexpected function for CTD in modulating gene expression.

Transition from initiation to promoter proximal pausing requires the CTD of RNA polymerase II

The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II Δ5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo. Here, we demonstrate that in the context of chromatin, Pol II Δ5 can bind to the c-myc promoter with the same efficiency as wild type Pol II. However, Pol II Δ5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged Δ5 or wildtype Pol II revealed a single, highly mobile Pol II Δ5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex.

Role of the Mammalian RNA Polymerase II C-Terminal Domain (CTD) Nonconsensus Repeats in CTD Stability and Cell Proliferation

The C-terminal domain (CTD) of mammalian RNA polymerase II (Pol II) consists of 52 repeats of the consensus heptapeptide YSPTSPS and links transcription to the processing of pre-mRNA. The length of the CTD and the number of repeats diverging from the consensus sequence have increased through evolution, but their functional importance remains unknown. Here, we show that the deletion of repeats 1 to 3 or 52 leads to cleavage and degradation of the CTD from Pol II in vivo. Including these repeats, however, allowed the construction of stable, synthetic CTDs. To our surprise, polymerases consisting of just consensus repeats could support normal growth and viability of cells. We conclude that all other nonconsensus CTD repeats are dispensable for the transcription and pre-mRNA processing of genes essential for proliferation.

A new mRNA polymerase

Mitochondria share a version of their RNA polymerase with the nucleus, as shown by Julia Kravchenko, Igor Rogozin, Eugene Koonin (NIH, Bethesda, MD), and Peter Chumakov (Cleveland Clinic Foundation, Cleveland, OH). This new fourth mammalian nuclear RNA polymerase adds a new dimension to mRNA

Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.

Comparative TFIIS-mediated transcript cleavage by mammalian RNA polymerase II arrested at a lesion in different transcription systems

Upon prolonged arrest at a cyclobutane pyrimidine dimer (CPD), RNAPII can reverse-translocate, misaligning the 3′-end of the RNA from its active site. Transcription factor SII (TFIIS) is required for cleavage of the disengaged 3′-end and restoration of its correct positioning. We have previously shown in vitro that when RNAPII is arrested at a CPD, TFIIS-induced cleavage results in shortened transcripts. Here, we hypothesized that the pattern of transcript cleavage does not depend solely upon TFIIS itself, but also on some other general transcription factors (GTFs) and/or their effects on RNAPII. To test this hypothesis we compared three in vitro transcription systems which differ with respect to the mode of initiation and the requirement for GTFs. The first consisted of RNAPII and GTFs from rat liver, and required a eukaryotic promoter for initiation. The other two supported transcription in the absence of any GTFs or promoter sequences. In each case, a CPD on the transcribed strand was a complete block for RNAPII translocation. However, the effect of TFIIS on transcript cleavage varied. In the promoter-initiated system, distinct transcripts up to about 20 nucleotides shorter than the uncleaved original one were produced. In the other two systems, the transcripts were degraded nearly completely. Introduction of GTFs partially interfered with cleavage, but failed to reproduce the pattern of transcript lengths observed with the promoter-initiated system. Our results suggest that the extent of TFIIS-mediated transcript cleavage is a well-orchestrated process, depending upon other factors (or their effects on RNAPII), in addition to TFIIS itself.

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II.

Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.

Protein kinase CK2: a new view of an old molecular complex.

Protein kinase CK2 (formerly known as casein kinase II) has been viewed traditionally as a stable heterotetrameric complex, but new analytical techniques are bringing a different picture into focus. The transient nature of this complex has been highlighted by the elucidation of its structure. Furthermore, analysis of the spatiotemporal organization of individual CK2 subunits in living cells has shown that they are dynamic and that they integrate into different multimolecular assemblies. These new studies give an additional dimension to the challenge of determining the cellular regulation of this protein kinase.

Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II

8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation. Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell. As a result, several repair processes have evolved to remove this lesion from the genome. It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes. A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR. As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII). We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter. We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95%. Similarly, this lesion only transiently blocked transcription by RNAPII. However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG. When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase. Binding of the Saccharomyces cerevisiae MSH2–MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase. These results are discussed in the context of possible models for TCR.

Non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase: discovery of benzimidazole 5-carboxylic amide derivatives with low-nanomolar potency

Optimization of benzimidazole 5-carboxamide derivatives previously identified as specific inhibitors of the NS5B polymerase of the hepatitis C virus (HCV) has led to the discovery of potent analogues that inhibit the enzyme at low-nanomolar concentrations. Greater than 800-fold improvement in potency from the original lead structure was achieved through the combined effects of conformational rigidification, molecular size extension and the identification of previously unexploited interactions. Furthermore, these inhibitors retain specificity for HCV polymerase relative to other viral and mammalian RNA polymerases.

The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability.

The phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) has been shown to affect the initiation, and transition to elongation of the Pol II complex. The differential phosphorylation of serines within this domain coincides with the recruitment of factors important for pre-mRNA processing and transcriptional elongation. A role for tyrosine and threonine phosphorylation has yet to be described. The discovery of kinases that express a preference for specific residues within this sequence suggests a mechanism for the controlled recruitment and displacement of CTD-interacting partners during the transcription cycle. The last CTD repeat (CTD52) contains unique interaction sites for the only known CTD tyrosine kinases, Abl1/c-Abl and Abl2/Arg, and the serine/threonine kinase casein kinase II (CKII). Here, we show that removal or severe disruption of the last CTD repeat, but not point mutation of its CKII sites, results in its proteolytic degradation to the Pol IIb form in vivo, but does not appear to affect the specific transcription of genes. These results suggest a possible mechanism of transcription control through the proteolytic removal of the Pol II CTD.

Cisplatin-induced ubiquitination of RNA polymerase II large subunit and suppression of induction by 7-hydroxystaurosporine (UCN-01)

Exposure of cells to DNA-damaging agents induces hyperphosphorylation of the C-terminal domain (CTD) of mammalian RNA polymerase II (RNAP II) large subunit (LS); the hyperphosphorylated RNAP II is then ubiquitinated. The purpose of this study was to verify that cisplatin-induced RNAP II ubiquitination is transcription dependent in living cells and to determine whether 7-hydroxystaurosporine (UCN-01) inhibits the ubiquitination induced by cisplatin. Cisplatin at clinically achievable concentrations (2.5-10 micro M) induced the ubiquitination of RNAP II in exponentially growing A2780 human ovarian tumor cells; the effect was drug-dose and exposure-time dependent. Such induction, however, was not observed in colcemid-selected mitotic cells. When detergent extraction was applied, the ubiquitinated RNAP II was recovered in the detergent-insoluble fraction, indicating that the protein was tightly bound to DNA. In an in vitro transcription reaction that consists of nuclear extracts and an immobilized DNA template containing a site-specific cisplatin lesion, the elongating RNAP II that was stalled at a cisplatin lesion site on the template was targeted by ubiquitins. Together, our results indicate that the ubiquitination is associated with transcription-coupled repair. We previously showed that the Ser/The kinase-inhibitor UCN-01 inhibits nucleotide excision repair. Here, we further determined the effect of UCN-01 on the phosphorylation and ubiquitination of RNAP II LS in a whole-cell system. Immunoblotting results showed that UCN-01 suppressed the cisplatin-induced ubiquitination and the cisplatin-induced shift from the hypophosphorylated IIa to the hyperphosphorylated IIo, without affecting the basal levels of the IIo and IIa forms of the RNAP II CTD, suggesting that UCN-01 acts by suppressing cisplatin-mediated induction of the one or more kinases that is responsible for the conversion of the IIo that is important for ubiquitination.

Behavior of T7 RNA Polymerase and Mammalian RNA Polymerase II at Site-specific Cisplatin Adducts in the Template DNA

Transcription-coupled DNA repair is dedicated to the removal of DNA lesions from transcribed strands of expressed genes. RNA polymerase arrest at a lesion has been proposed as a sensitive signal for recruitment of repair enzymes to the lesion site. To understand how initiation of transcription-coupled repair may occur, we have characterized the properties of the transcription complex when it encounters a lesion in its path. Here we have compared the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We found that a single cisplatin 1,2-d(GG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to both polymerases. Furthermore, the efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link was similar in several different nucleotide sequence contexts. Interestingly, some blockage was also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link was located in the non-transcribed strand. Transcription complexes arrested at the cisplatin adducts were substrates for the transcript cleavage reaction mediated by the elongation factor TFIIS, indicating that the RNA polymerase II complexes arrested at these lesions are not released from template DNA. Addition of TFIIS yielded a population of transcripts up to 30 nucleotides shorter than those arrested at the lesion. In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair.

The repetitive C-terminal domain of RNA polymerase II: multiple conformational states drive the transcription cycle.

RNA polymerase (RNAP) II is a complex multisubunit enzyme responsible for the synthesis of mRNA in eukaryotic cells. The largest subunit contains at its C-terminus a unique domain, designated the CTD, comprised of tandem repeats of the consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). This repeat occurs 52 times in mammalian RNAP II. The CTD is subject to extensive phosphorylation at specific points in the transcription cycle by distinct CTD kinases that phosphorylate certain positions within the consensus repeat. The level and pattern of phosphorylation is determined by the concerted action of CTD kinases and CTD phosphatases. The highly dynamic modification by multiple CTD kinases and phosphatases generate distinct conformations of the CTD that facilitate the recruitment of specific macromolecular assemblies to RNAP II. These CTD interacting proteins influence formation of a preinitiation complex at the promoter and couple processing of the primary transcript to the elongation complex.

Effects of Endogenous DNA Base Lesions on Transcription Elongation by Mammalian RNA Polymerase II: IMPLICATIONS FOR TRANSCRIPTION-COUPLED DNA REPAIR AND TRANSCRIPTIONAL MUTAGENESIS

The blockage of transcription elongation by RNA polymerase II (pol II) is thought to be a trigger for transcription-coupled repair in the pathway of nucleotide excision repair. Purified pol II and oligo(dC)-tailed templates containing a single non-bulky DNA lesion on the transcribed strand such as an apurinic/apyrimidinic (AP) site, uracil, or 8-oxoguanine (8-oxoG) were used for transcription elongation assays. In this system pol II could bypass both the AP site and uracil without pausing and insert cytosine opposite the AP site and either guanine or adenine opposite to uracil. Thus, the AP site on the DNA templates could lead to correct transcription only if depurination at guanine occurred, whereas uracil generated either the correct transcriptional product or an incorrect one with a G:C to A:T transition. In the case of 8-oxoG, pol II stalled at the lesion, but sometimes bypassed it and inserted a cytosine residue or the incorrect adenine residue leading to a G:C to T:A transversion. These findings indicate that 8-oxoG lesions caused a blockage of transcription elongation and/or the misincorporation of a ribonucleotide by pol II, implying the initiation of transcription-coupled repair of 8-oxoG and/or transcriptional mutagenesis.

Template Switching by RNA Polymerase II In Vivo: Evidence and Implications from a Retroviral System

Transfection of retrovirus packaging cells with linear DNA from a retroviral vector missing the 3′ long terminal repeat (3′ LTR) results in production of infectious virus. Analysis of the newly formed proviruses indicates that restoration of the 3′ LTR sequences necessary for reverse transcription and integration occurred due to end-to-end template switching by mammalian RNA polymerase II (RNAP II) in the packaging cells. These observations argue that RNAP II can utilize double-strand breaks and gaps in DNA to generate “recombinant” transcripts in vivo and suggest a mechanism for mutation and recombination of retroviruses.

A kinetic framework for a mammalian RNA polymerase in vivo.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

A single 8,5′-cyclo-2′-deoxyadenosine lesion in a TATA box prevents binding of the TATA binding protein and strongly reduces transcription in vivo

8,5′-Cyclo-2′-deoxypurine (cPu) lesions result from the action of the hydroxyl radical on DNA. These lesions represent a unique class of oxidative DNA lesions in that they are repaired by the nucleotide excision repair (NER) pathway but not by base excision repair (BER) or direct repair. Previous work has shown that cyclopurines can block mammalian DNA and RNA polymerases. Thus, these lesions are of interest because of their potential role in the neurodegeneration as well as internal cancers observed in patients with xeroderma pigmentosum (XP) who lack the capacity to carry out NER. In the present work, we found that the S-isomer of 8,5′-cyclo-2′-deoxyadenosine (cA) can prevent binding of the TATA binding protein (TBP) to the TATA box from the CMV promoter. To assess the functional importance of this effect in living cells, we transfected constructs containing a single cA in the CMV TATA box into XP cells to determine the effect of the lesion on gene expression in vivo. Using this approach, we found that the lesion reduced gene expression by approximately 75%. This effect was comparable to the effect of an inactivating mutation of the TATA box in the same promoter. These findings identify an additional biological effect of cyclopurine lesions in mammalian cells, which is the ability to interfere with transcription by preventing transcription factor binding to cognate recognition sequences. In addition, the approach we used in this study represents a novel method for assessing the effects of DNA lesions in non-transcribed sequences on gene expression in living cells.

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

The carboxyl-terminal domain (CTD) of the largest subunit of mammalian RNA polymerase II (RNAP II) consists of 52 repeats of a consensus heptapeptide and is subject to phosphorylation and dephosphorylation events during each round of transcription. RNAP II activity is regulated during the cell cycle and cell cycle-dependend changes in RNAP II activity correlate well with CTD phosphorylation. In addition, global changes in the CTD phosphorylation status are observed in response to mitogenic or cytostatic signals such as growth factors, mitogens and DNA-damaging agents. Several CTD kinases are members of the cyclin-dependent kinase (CDK) superfamily and associate with transcription initiation complexes. Other CTD kinases implicated in cell cycle regulation include the mitogen-activated protein kinases ERK-1/2 and the c-Abl tyrosine kinase. These observations suggest that reversible RNAP II CTD phosphorylation may play a key role in linking cell cycle regulatory events to coordinated changes in transcription. J. Cell. Physiol. 190: 160–169, 2002. © 2002 Wiley-Liss, Inc.