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In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co-activator. Here, we employed single-particle cryo-electron microscopy (cryo-EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ~19 A. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf-Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII-TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA-binding cleft, in agreement with cross-linking and biochemical mapping. Gdown1's binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated transcription.

The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1α-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfβ, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function.

Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pol II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function., National Human Genome Research Institute (U.S.) (Grant HG002668-05)

Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (noninitiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.

SignificanceCyclopurines are bulky oxidative DNA lesions that strongly block RNA polymerase II (Pol II) to interfere with gene transcription and replication in mammalian cells. Cells developed a mechanism enabling slow transcriptional bypass of the cyclopurines. Similar to translesion synthesis by DNA polymerases, lesion bypass by Pol II can be highly mutagenic, leading to transcription errors that are reminiscent of the A rule. We elucidated the mechanism and determined the domain in Pol II responsible for error-free and error-prone lesion bypass. We also identified a positive role of mammalian factor TFIIF in lesion-bypass stimulation. Strikingly, Pol II uses a similar strategy for negotiation with different DNA lesions, such as cyclopurines, pyrimidine dimers, cisplatin, and abasic sites, to reduce the burden of DNA damage on genome stability.In human cells, the oxidative DNA lesion 8,5′-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5′) next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5′-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene- and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediator-dependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.

The INrf2 (Keap1)–Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3–Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCε is essential for INrf2–Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCε, knockdown of PKCε and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2–Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2–Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCε and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCε leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCε.

Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 Å resolution. The validity of the phases was shown by close similarity of a 6 Å electron density map to a 16 Å molecular envelope of the polymerase from electron crystallography. Comparison of the 6 Å X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8–18 residues upstream of the 3′-end in a tunnel.

Minimal templates were devised for the efficient generation of yeast RNA polymerase II transcription elongation complexes. A 33-base pair DNA with a 15-residue dC tail at one 3'-end supported the formation of a complex containing the polymerase paused at nucleotide 11 of the duplex region and an RNA of 14-16 residues. The same template could yield an arrested complex with the enzyme at nucleotide 13-15 and RNA of 15-17 residues. These complexes were stable for at least a week under various conditions and could be resolved by gel electrophoresis or purified by ion exchange chromatography. The purified paused complex formed crystals capable of x-ray diffraction to 3.5 A resolution. The complex remained active in the crystal and, in the presence of nucleoside triphosphates, could efficiently extend the transcript in situ.

Free and elongating (DNA-bound) forms of RNA polymerase II were separated from yeast. Most cellular polymerase II was found in the elongating fraction, which contained all enzyme phosphorylated on the C-terminal domain and none of the 15-subunit mediator of transcriptional regulation. These and other findings suggest that mediator enters and leaves initiation complexes during every round of transcription, in a process that may be coupled to C-terminal domain phosphorylation.

The INrf2 (Keap1)-Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3-Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCε is essential for INrf2-Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCε, knockdown of PKCε and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2-Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2-Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCε and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCε leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCε.

The INrf2 (Keap1)–Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3–Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCe is essential for INrf2–Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCe, knockdown of PKCe and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2–Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2–Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCe and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCe leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCe.

Single-molecule studies of RNA polymerase II (RNAP II) require high yields of transcription elongation complexes (TECs) with long DNA tethers upstream and downstream of the TEC. Here we report on a robust system to reconstitute both yeast and mammalian RNAP II with an efficiency of ~80% into TECs that elongate with an efficiency of ~90%, followed by rapid, high-efficiency tripartite ligation of long DNA fragments upstream and downstream of the reconstituted TECs. Single mammalian and yeast TECs reconstituted with this method have been successfully used in an optical-trapping transcription assay capable of applying forces that either assist or hinder transcript elongation.

Acetyl- and butyrylcholinesterases (ACHE, BuChE) from various species differ in their substrate specificities and sensitivities to a wide range of inhibitors, yet display conserved sequence, structure and catalytic properties. To determine features that confer these selective properties, residues 58 through 133 of recombinant human BuChE were replaced with the corresponding sequence from human ACHE. The replaced region (>60% identity) spans the Asp70 residue, important for ligand interactions, and the choline binding site, and introduces differences of charge and hydrophobicity in the outer rim and on the surface of the active site gorge. Expressed in microinjected Xenopus laevis oocytes, the resultant chimera retained the catalytic activity, substrate specificity and the Km value toward butyrylthiocholine characteristic of BuChE. Further, it did not acquire substrate inhibition, which is unique to ACHE, although it lost the property of substrate activation, characteristic of BuChE. Moreover, the chimera resembled BuChE in its sensitivity to succinylcholine and physostigmine, but acquired the AChE-like sensitivity to echothiophate and iso-OMPA, and displayed an intermediate pattern of inhibition, more similar to that of AChE than of BuChE, toward bambuterol, dibucaine and BW284C51. These findings demonstrate that the exchanged residues are involved in inhibitor recognition, but not in substrate distinction and in direct catalysis. Furthermore, substrate interaction with the exchanged domain may mediate structural changes leading to substrate activation in BuChE and inhibition in ACHE. The two AChE-specific aromatic tyrosine residues positioned near Asp70 within this region are hence implicated in the peripheral anionic site of cholinesterases, which is involved in the recognition of various ligands.

Yael Loewenstein, Averell Gnatt, Lewis F. Neville, Haim Zakut and Hermona Soreq: Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1993, 17(6): 905–926. 1. 1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. 4. Thus, it was found that asp70→gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425→Pro with gly70 gives rise to the “atypical” butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimer's and Parkinson's disease, glaucoma and myasthenia gravis.

Acetyl- and butyrylcholinesterase (ACHE, BCHE) from evolutionarily distant species display a high degree of primary sequence homology and have biochemically similar catalytic properties, yet they differ in substrate specificity and affinity for various inhibitors. The biochemical information derived from analyses of ACHE and BCHE from human, Torpedo, mouse, and Drosophila, as well as that from the recombinant forms of their natural variants and site-directed mutants, can currently be re-examined in view of the recent X-ray crystallography data revealing the three-dimensional structure of Torpedo ACHE. The picture that emerges deepens the insight into the biochemical basis for choline ester catalysis and the complex mechanism of interaction between cholinesterases and their numerous ligands.

Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center.

Acetaldehyde, the first metabolite of ethanol, reacts with DNA to form adducts, including N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG). Although the effects of N(2)-Et-dG on DNA polymerases have been well studied, nothing is known about possible effects of this lesion on transcription by RNA polymerases (RNAPs). Using primer extension assays in vitro, we found that a single N(2)-Et-dG lesion is a strong block to both mammalian RNAPII and two other multisubunit RNAPs, (yeast RNAPII and Escherichia coli RNAP), as well as to T7 RNAP. However, the mechanism of transcription blockage appears to differ between the multisubunit RNAPs and T7 RNAP. Specifically, all three of the multisubunit RNAPs can incorporate a single rNTP residue opposite the lesion, whereas T7 RNAP is essentially unable to do so. Using the mammalian RNAPII, we found that CMP is exclusively incorporated opposite the N(2)-Et-dG lesion. In addition, we also show that the accessory transcription factor TFIIS does not act as a lesion bypass factor, as it does for other nonbulky DNA lesions; instead, it stimulates the polymerase to remove the CMP incorporated opposite the lesion by mammalian RNAPII. We also include models of the N(2)-Et-dG within the active site of yeast RNAPII, which are compatible with our observations.

The "atypical" allelic variant of human butyrylcholinesterase (BuChE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BuChE enzyme. A single nucleotide substitution conferring a change of aspartate-70 into glycine was recently reported in the CHE gene encoding BuChE from several individuals having the "atypical" BuChE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine-425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BuChE, three transcription constructs were engineered with each of these substitutions alone or both of them together. Xenopus oocyte microinjection of normal or mutated synthetic BuChEmRNA transcripts was employed in conjunction with biochemical analyzes of the resultant recombinant BuChE variants. The presence of the Gly-70 mutation alone was found to render the enzyme resistant to 100 microM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the "normal," Asp-70 containing BuChE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly-70 BuChE failed to be reactivated by 10 mM of the cholinesterase-specific oxime pyridine 2-aldoxime methiodide (2-PAM); a concentration restoring about 50% of activity in the "normal" Asp-70 recombinant enzyme. The Pro-425 mutation alone had no apparent influence on BuChE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly-70 mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.

BackgroundA common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture.MethodsRNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines.ResultsKnockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death.ConclusionAlthough transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery.

One common way to examine toxicity in vitro is to measure the effect on cell viability. In one such assay, the 3-(4,5- dimethylthiazol-2-yl)-5-(3 -carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTS reagent is bioreduced to a formazan product in living cells. Various cell lines may have differing abilities to reduce MTS; therefore, standardization should be carried out for each one. To optimize the assay, the toxicity of MTS, the linear range for signal versus cell number, and a method of background noise reduction were determined. The MTS reagent decreased cell number after 25 hr. The linear range for the neuronal SK-N-Sll, esophageal KYSE-30, breast MCF-7, and cervical HeLa cell lines were established. Finally, media containing no phenol red significantly reduced background noise compared to media with phenol red.

Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1–CTD interaction has remained ambiguous because two different modes have been proposed: the “dockingsite” model versus the “distributive” mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously.

RNA polymerase II is the eukaryotic enzyme that transcribes all the mRNA in the cell. Complex mechanisms of transcription and its regulation underlie basic functions including differentiation and morphogenesis. Recent evidence indicates the process of RNA chain elongation as a key step in transcription control. Elongation was therefore expected and found to be linked to human diseases. For these reasons, major efforts in determining the structures of RNA polymerases from yeast and bacteria, at rest and as active enzymes, were undertaken. These studies have revealed much information regarding the processes involved in transcription. Eukaryotic RNA polymerases and their homologous bacterial counterparts are flexible enzymes with domains that separate DNA and RNA, prevent the escape of nucleic acids during transcription, allow for extended pausing or “arrest” during elongation, allow for translocation of the DNA and more. Structural studies of RNA polymerases are described below within the context of the process of transcription elongation, its regulation and function.

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 Å resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3′ end of the RNA in the nucleotide addition site. The 3′ end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5′-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of “switches” at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein–nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, “abortive cycling” during transcription initiation, and RNA and DNA translocation during transcription elongation.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography. Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure. Details are indicative of a roughly 90° bend of the DNA between upstream and downstream regions. The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested.

The purpose of the proposed research is to provide a structural basis for understanding the mechanism of transcription, its regulation, and altered regulation as occurs in tumor cells. The goal of the proposed research is to determine the X-ray structure of RNA Polymerase II in the midst of transcribing RNA from a DNA template. During the first year of funding, template DNA was shortened to a minimal size without affecting the stability of the paused complex or its efficiency of formation. It was determined that previous platelike crystals are sensitive to physical manipulation, soaking in cryoprotectant or soaking with heavy metal clusters. Crystallization trials of polymerase resulted in new non-platelike crystal forms allowing for the collection of a preliminary data set to 7.5 Angstroms. This is the best elongation complex data set, since previous platelike crystals had mosaic spreads over 1 degree. Recently new crystallization conditions allowed the growing of crystals in cryoprotectant and the generation of yet new crystal forms. Growing in cryoprotectant is a major accomplishment considering the sensitivity of the crystals to manipulations.

X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity., Author Summary Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by side effects of unknown origin. Here we show in biochemical and cell-based studies that antiviral ribonucleotide analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Analogues that terminate RNA synthesis by viral RNA polymerases also inhibit these cellular RNA polymerase. Importantly, the TFIIS proofreading activity of Pol II is capable of excising these analogues from Pol II transcripts. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

Gene amplification occurs frequently in tumour tissues yet is, in general, non-inheritable. To study the molecular mechanisms conferring this restraint, we created transgenic mice carrying a human butyrylcholinesterase (BCHE) coding sequence, previously found to be amplified in a father and son. Blot hybridization of tail DNA samples revealed somatic transgene amplifications with variable restriction patterns and intensities, suggesting the occurrence of independent amplification events, in 31% (11/35) of mice from the FII generation but in only 3.5% (2/58) of the FIII and FIV generations. In contrast, > 10-fold amplifications of the BCHE transgene and the endogenous acetylcholinesterase and c-raf genes appeared in both testis and epididymis DNA from > 80% of FIII mice. Drastic, selective reductions in testis BCHEmRNA but not in actin mRNA were detected by the PCR amplification of testis cDNA from the transgenic mice, and apparently resulted in the limited transmission of amplified genes. The testicular amplification of the BCHE transgene may potentially represent a general phenomenon with clinical implications in human infertility.

Multiple findings implicate acetylcholine with sperm functioning 1,2 and acetyl-and butyrylcholinesterase activities (ACHE, BCHE) were observed in mammalian sperm cells and during oocyte development 1–3. In vivo amplification of the human BCHE gene was first found in a father and son exposed to cholinesterase inhibitors 4, but it remained unclear whether the amplified DNA was transmitted as such from father to son or whether the amplification phenomenon re-occurred in germ cells, particularly during male meiosis or sperm differentiation.

The two classes of human cholinesterases (CHEs), acetylcholinesterase(acetylcholine acetyl hydrolase, ACHE, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acyl hydrolase, BCHE, EC 3.1.1.8) are highly homologous proteins capable of rapidly hydrolyzing choline estersl. Despite their similar mechanisms of action, they differ in substrate specificity and sensitivity to various inhibitors1,2. Recent advances including cloning,3–6 expression,5,7–10 and 3 dimensional structural analysis of members of the CHE superfamily,11,12 now enable the dissection of functional domains thereof. Within these domains, key amino acids are found which may be implicated in catalysis or in binding of various ligands. The disclosing of such key residues could lead to designing of novel therapeutic agents as well as to the unravelling of the molecular mechanisms underlying the functioning of ChEs.

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5′ fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3′ end of BuChEcDNA from nervous system tumors and which has been mapped byin situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-coding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringentin situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family of highly similar proteins encoded by distinct, nonhomologous genes.

The human Cholinesterase genes and their protein products have been the focus of intensive research for many years (for comprehensive reviews see Whittaker, 1986; 1Rakonczay and Brimijoin, 1988; and Soreq and Zakut, 1990) because of the physiological function attributed to these enzymes, which are both capable of hydrolyzing the neurotransmitter acetylcholine. Genetic linkage evidence indicated that two distinct genes, designated ACHE and CHE, encode the two principal forms of cholinesterases, acetylcholinesterase (acetylcholine acetyl hydrolase, AChE, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, BuChE, EC 3.1.1.8) which differ in their substrate specificities and sensitivities to selective inhibitors. The toxic effects of organophosphorous (OP) poisons, such as common insecticides or nerve gases, are generally attributed to their specific inhibition of cholinesterases, interfering with cholinergic neurotransmission. OP inhibition of cholinesterases occurs through a covalent interaction of the OP compounds with a serine residue in the active esteratic site (Koelle, 1972). However, detailed structure-function relationships in this family of enzymes have been hampered by the difficulties in purifying mammalian cholinesterases.

Attempts to restore cholinergic deficits in Alzheimer’s disease patients involve the use of various drugs inhibiting cholinesterases (CHEs; Bartus et al., 1982). In addition, CHE inhibitors are clinically employed in the treatment of other common syndromes, including Parkinson’s disease, myasthenia gravis and multiple sclerosis (Taylor, 1990). The efficacy and specificity of such drugs on the one hand, and their toxicity factor on the other, largely depend on their inhibitory effects on CHEs in the treated individuals. Therefore, updated evaluation methods for these parameters should be valuable to pharmacological research focused on the development and use of cholinergic drugs.

Structure-function relationships of recombinant human butyrylcholinesterase (CHE) variants were investigated by Xenopus oocyte microinjection. A Ser-425 to Pro-425 mutation failed to modify ligand binding properties. In contrast, Asp-70 to Gly-70 substitution significantly reduced CHE binding capacity for succinylcholine and specific inhibitors, demonstrating Asp-70 as a key anionic site component for certain ligands. Furthermore, the presence of both mutations rendered CHE totally resistant to succinylcholine and dibucaine inhibition, while all mutant proteins bound butyrylthiocholine, benzoylcholine, and propionylcholine normally. These findings imply structural interactions between the conserved Asp-70 and Ser-425 regions in cholinesterases and suggest the contribution of additional electronegative amino acids to anionic site binding.

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.

Cholinesterases (ChEs) are highly polymorphic proteins, capable of rapidly hydrolyzing the neurotransmitter acetylcholine and involved in terminating neurotransmission in neuromuscular junctions and cholinergic synapses. In an attempt to delineate the structure and detailed properties of the human protein(s) and the gene(s) coding for the acetylcholine hydrolyzing enzymes, a human cDNA coding for ChE was isolated by use of oligodeoxynucleotide screening of cDNA libraries. For this purpose, a method for increasing the effectiveness of oligonucleotide screening by introducing deoxyinosine in sites of codon ambiguity and using tetramethyl-ammonium salt washes to remove false-positive hybrids was employed. The resulting isolated 2.4-kilobase (kb) cholinesterase cDNA sequences encode for the entire mature secretory protein, preceded by an N-terminal signal peptide. The human ChE primary sequence shows almost no homology to other serine hydrolases, with the exception of a hexapeptide at the active site. In contrast, it displays extensive homology with acetylcholinesterase form Torpedo californica and Drosophila melanogaster as well as with bovine thyroglobulin. These extensive homologies probably suggest the need of the entire coding sequence for the physiological function(s) fulfilled by the enzyme and further suggest a common, unique, ancestral gene for these cDNAs. In turn, the cDNA was used as a probe to isolate genomic DNA sequences for the 5'-region of the human ChE gene. The genomic DNA fragment encoding part of the 5'-region of ChEcDNA was detected by DNA blot hybridization, enriched 70-fold by gel electrophoresis and electroelution, cloned in lambda phage and isolated. Sequencing of the cloned DNA revealed that it did indeed include part of the 5'-region of ChEcDNA, starting at an adjacent 5'-position to the nucleotides coding for the initiator methionine, and ending with an EcoRI restriction site inherent to the ChEcDNA sequence. The isolated fragment of the human cholinesterase gene is currently employed to complete the structural characterization of this and related genes.