Human RNA Polymerase Research Articles

Co-expression of multiple subunits enables recombinant SNAP C assembly and function for transcription by human RNA polymerases II and III

Human small nuclear (sn) RNA genes are transcribed by either RNA polymerase II or III depending upon the arrangement of their core promoter elements. Regardless of polymerase specificity, these genes share a requirement for a general transcription factor called the snRNA activating protein complex or SNAP C. This multi-subunit complex recognizes the proximal sequence element (PSE) commonly found in the upstream promoters of human snRNA genes. SNAP C consists of five subunits: SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. Previous studies have shown that a partial SNAP C composed of SNAP190 (1–514), SNAP50, and SNAP43 expressed in baculovirus is capable of PSE-specific DNA binding and transcription of human snRNA genes by RNA polymerases II and III. Expression in a baculovirus system yields active complex but the concentration of such material is insufficient for many bio-analytical methods. Herein, we describe the co-expression in Escherichia coli of a partial SNAP C containing SNAP190 (1–505), SNAP50, SNAP43, and SNAP19. The co-expressed complex binds DNA specifically and recruits TBP to U6 promoter DNA. Importantly, this partial complex functions in reconstituted transcription of both human U1 and U6 snRNA genes by RNA polymerases II and III, respectively. This co-expression system will facilitate the functional characterization of this unusual multi-protein transcription factor that plays an important early role for transcription by two different polymerases.

1033. Tightly Controlled and Reversible Knockdown of Gene Expression Using Lentiviral Vectors Encoding Tetracycline-Dependent Repressors or Transactivators

RNAi accomplished in mammalian cells by the introduction of small interfering RNAs (siRNAs) is a powerful emerging tool for reverse genetic studies. To control siRNA-mediated gene knockdown both spatially and temporally, we undertook the development of HIV-1-based lentiviral vector systems for the conditional, doxycycline (Dox) dependent production of short hairpin RNAs (shRNAs) in mammalian cells from modified human RNA polymerase III (Pol III) promoters. Our results showed that expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or a second- generation Tet-responsive promoter element (TRE) upstream ofthe promoter was tight and reversible in the presence of a modified Tet-repressor (TetR).

RNA Polymerase II Blockage by Cisplatin-damaged DNA: STABILITY AND POLYUBIQUITYLATION OF STALLED POLYMERASE

The consequences of human RNA polymerase II (pol II) arrest at the site of DNA damaged by cisplatin were studied in whole cells and cell extracts, with a particular focus on the stability of stalled pol II and its subsequent ubiquitylation. Site-specifically platinated DNA templates immobilized on a solid support were used to perform in vitro transcription in HeLa nuclear extracts. RNA elongation was completely blocked by a cisplatin intrastrand cross-link. The stalled polymerase was quite stable, remaining on the DNA template in nuclear extracts. The stability of pol II stalled at the site of cisplatin damage was also observed in live cells. A cell fractionation experiment using cisplatin-treated HeLa cells revealed an increased level of chromatin-associated pol II proteins following DNA damage. The stalled polymerase was transcriptionally active and capable of elongating the transcript following chemical removal of platinum from the template. Transcription inhibition by alpha-amanitin in vitro enhanced pol II ubiquitylation at ubiquitin residues Lys-6, Lys-48, and Lys-63. In live cells expressing epitope-tagged ubiquitin mutants, several ubiquitin lysines also participated in pol II ubiquitylation following DNA damage. Cisplatin treatment triggered ubiquitylation-mediated pol II degradation in HeLa cells, which could be prevented by the proteasomal inhibitor MG132. Fractionation of pol II from cells co-treated with MG132 and cisplatin indicated that the undegraded ubiquitylated polymerase was mostly unbound or only loosely associated with chromatin. These data are consistent with a model in which only a fraction of pol II, ubiquitylated in response to cisplatin damage of DNA, dissociates from the sites of platination. This altered polymerase is rapidly destroyed by proteasomes.

A Multivariate Analysis of Human Promoter Sequences.

The human genome has been estimated to contain tens of thousands of genes. Of these, the promoters have been experimentally verified for almost two thousand. We have examined the DNA sequences just up-stream of the transcription start site, a region which includes the TATA box. Genetic control sites, such as promoters, often have a characteristic consensus sequence, but the variation about a given consensus sequence has received little attention. Sequence variations may be related to functional differences amongst the control sites. Principal components analysis has been chosen because of its generality and the variety of phenomena which it reveals. Promoter sequences were considered because of the large number available and their importance in gene expression. The sequences of the 1977 promoters recognised by human RNA polymerase II were obtained from the Eukaryotic Promoter Database. Many of these promoters are of interest in oncology and the database includes sequences for growth factors (e.g. GM-CSF, interleukins), oncogenes and tumour viruses among others. Sub-sequences of 25 bases centred on position −13 relative to the transcription start site were extracted. Two bits were used to encode each base (a=11, c=00, g=10 and t=01) and the covariance matrix of the resulting 50 variables was determined. The eigenvalues and eigenvectors of the covariance matrix were calculated. All calculations were carried out by computer using MS-Excel and SYSTAT 11. The eigenvalues of the covariance matrix ranged from 0.571 down to 0.133. The eigenvectors were used to calculate principal components. Thus 50 more or less correlated variables were transformed into 50 uncorrelated variables with the same total variance. The sequences were sorted according to the principal components to reveal which features were associated with the most variation amongst the sequences. When the covariances among the coded sequences were calculated many associations were found, for example, a purine at position 15 was associated with a purine at position 16, and a purine at position 19 with a G or C at position 20. Although these correlations individually were not especially strong, together they were a notable feature of the set of sequences. The consensus sequence was observed to be agggg ggggg ggc(g/c)c ggggg gcgcc. A principal components analysis enabled the promoters to be identified which differed most (in opposite directions) from the consensus sequence, taking account of the correlations. Nearly all the elements of the first eigenvector were of alternating sign; thus the first principal component separated promoters which were rich in G from those rich in T. Almost all elements of the second eigenvector were positive, so the second principal component distinguished promoters rich in A from those rich in C. There was a remarkable concentration of promoters from genes for interleukins or IL repressors with large values for the second principal component:- IL1A, IL2, IL4, IL6-2, IL2RA1, IL2RA2 and IL8RB were in positions 160, 43, 14, 158, 131, 101 and 158 (out of 1977) respectively. The variation in the sequence of promoters about their consensus sequence is seen not to be random but to display detectable patterns. Correlations were found to be frequent within the promoter sequences considered here; in the absence of correlations all the eigenvalues would have been equal. The major principal components separated promoters with markedly different sequences. It is to be expected that the other principal components would yield further separations.

Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II.

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) form a heterodimer that protrudes from the 10-subunit core of the enzyme. We have obtained crystals of the human Rpb4/Rpb7 heterodimer and determined the structure to 2.7 Å resolution. The presence of putative RNA-binding domains on the Rpb7 subunit and the position of the heterodimer close to the RNA exit groove in the 12 subunit yeast polymerase complex strongly suggests a role for the heterodimer in binding and stabilizing the nascent RNA transcript. We have complemented the structural analysis with biochemical studies directed at dissecting the RNA-binding properties of the human Rpb4/Rpb7 complex and that of the homologous E/F complex from Methanocaldococcus jannaschii. A number of conserved, solvent-exposed residues in both the human Rpb7 subunit and the archaeal E subunit have been modified by site-directed mutagenesis and the mutants tested for RNA binding by performing electrophoretic mobility shift assays. These studies have identified an elongated surface region on the corresponding face of both subunit E and Rpb7 that is involved in RNA binding. The area spans the nucleic acid binding face of the OB fold, including the B4–B5 loop, but also extends towards the N-terminal domain.

Mutagenic properties of ribonucleotide analogues in reverse transcription with HIV and AMV reverse transcriptases

Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA. The incorporation ratio of dGTP to dATP opposite each analogue was measured to estimate the potential for inducing U-to-C mutations. The potentials may be rC(oh) > rC(mo) > rP for both reverse transcriptases. It might be possible to induce mutations in the retroviral genomes and to develop a new antiviral therapy, if these analogues are incorporated by a human RNA polymerase.

The antiproliferative agent MLN944 preferentially inhibits transcription

MLN944 is a novel compound currently being codeveloped by Millennium Pharmaceuticals and Xenova Ltd. as a cancer therapeutic and is in a phase I clinical trial for solid tumors. Although MLN944 was originally proposed to function as a topoisomerase I and II inhibitor, more recent data has shown that it is a DNA-intercalating agent that does not inhibit the catalytic activity of topoisomerase I or II. We show here that MLN944 inhibits incorporation of radiolabeled precursors into RNA preferentially over incorporation into DNA and protein in HCT116 and H460 cells. To determine if MLN944 inhibits transcription, a human RNA polymerase II in vitro transcription system was used. MLN944 inhibited initiation when added before or after the formation of preinitiation complexes and inhibited elongation at higher concentrations. The preferential inhibition of initiation differentiates MLN944 from actinomycin D, which more strongly inhibits elongation. Transcription of all RNA polymerases was inhibited in nuclei isolated from HeLa cells treated with low concentrations of MLN944. Our data are consistent with transcription as the target of the potent cytotoxic effects of MLN944.

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.

951. Evaluation of HSV-Mediated Alzheimer Gene Therapy Vectors Using Novel Alzheimer Mouse Model: Applications of siRNA and Neprilysin Vectors to Adjust the Metabolic Imbalance of Aâ in Alzheimer's Disease

Alzheimer's disease (AD) is the most common type of dementia occurring in mid- to late-life, affecting more than 4 million individuals in the United States alone. The amyloid--protein (A), which is generated from the amyloid precursor protein (APP) by - and -secretase cleavage, is a major component of pathological plaques that accumulate in AD brains. The majority of AD patients develop the disease due to over-secretion or faulty clearance of A peptide. Thus down-regulation of the expression of A or stimulation of the A clearance process may ameliorate or potentially cure the devastating disease. As we previously have presented, we have developed replication-defective HSV-mediated gene therapy vectors to favorably adjust the metabolic imbalance of A. The first vector (QAPPi), which carries an siRNA expression cassette targeting APP gene under control of human H1 RNA polymerase III promoter, significantly lowered APP protein level compared to controls in MOI-dependent manner in vitro. The second vector (QNep), which carries Neprilysin, potent A degrading protease, produced a five fold decrease on A secretion compared to cells infected with an isogenic control virus. These results support the hypothesis that HSV-mediated APP-siRNA or Neprilysin expression may reduce A levels in vivo. To test this hypothesis in vivo, we have developed a novel in vivo mouse model using a lentivirus delivering a familial AD mutant form of APP gene (Swedish mutation). The targeted viral delivery system for modeling neurodegenerative diseases has several advantages over germline transgenic mice such as temporal control of transgene expression to avoid any developmental effect and to study aging effect, spatial control of transgene expression to target brain region associated with specific disease, and ability to deliver several transgenes. We are currently investigating the in vivo efficacy of the QAPPi and QNep vectors in the APP lentivirus model.

Computational Search for Potential Posttranslational Modification Sites in Human RNA Polymerase III Subunits

The transcription of small stable non-translated RNA genes (class III genes), directed by RNA polymerase III, is strictly regulated in accordance to physiological state of the cell (growth rate, cell cycle stage, apoptosis, etc.) Post-translational modifications of the polymerase may play the important role in class III gene transcription regulation. Using computational programs searching for potential post-translational modifications sites in proteins (MotifScan, NetPhos 2.0, and Yin-Yan 1.2), possible sites of phosphorylation were identified in all 17 subunits of human RNA polymerase III, and possible sites of reciprocal phosphorylation and glycosilation ("yin-yan" sites) - in 13 subunits. Among the identified sites -17 sites of phosphorylation in seven subunits are conservative in human, Saccharomyces cerevisiae and Schizosaccharomyces pombe, including two "yin-yan" sites in two subunits. The data obtained can be used for experimental identification of RNA polymerase III modification sites in vivo in cells being in different physiological states.

381. Methods for Optimizing Lentiviral Vector-Mediated Gene Suppression by RNA Interference

Short interfering (si)RNA methods have the potential for specific suppression of target genes, but finding the optimal siRNA target sequence and evaluating the persistence of gene knockdown are challenges which hinder progress. We have developed a strategy for vector-based screening of multiple siRNA sequences against target genes in mammalian cells. The steps are as follows: 1) creation of a fusion construct of the gene of interest (GOI) and a fluorescent reporter such as GFP at either the N-or C-terminus, 2) stable expression of the GOI-GFP fusion in a mammalian cell line, 3) transfection of vector plasmids encoding templates for short-hairpin (sh)RNAs, under the transcriptional control of a human RNA polymerase III (H1) promoter, targeted against several GOI sequences in the stable cell line, 4) quantification of the GOI knockdown by flow cytometric analysis for diminished GFP fluorescence. Quantification of expression by this method permits not only the selection of the most suppressive sequences but also those capable of resulting in intermediate levels of suppresion and hence intermediate phenotypic outcomes. In a modification of step 3, the shRNA expression cassettes are subcloned into a lentiviral vector system permitting transduction of stably modified cells thereby enabling the evaluation of persistence of the suppressive effect after multiple subcultures.

Dynamic Error Correction and Regulation of Downstream Bubble Opening by Human RNA Polymerase II

The nucleotide triphosphate (NTP)-driven translocation hypothesis posits that NTP substrates bind to templated DNA sites prior to translocation into the active site. By using millisecond phase kinetics, we demonstrate this prediction in three different ways. First, we show that, in the presence of the translocation blocker alpha-amanitin, NTPs (but not deoxynucleotide triphosphate [dNTPs]) templated at downstream sites (i + 2 and i + 3) dislodge an active site (i + 1) NTP, which was otherwise fated to complete bond synthesis. Second, we show that NTPs templated at i + 2 and/or i + 3 downstream sites suppress misincorporation errors. Third, we show that NTPs templated at downstream sites stabilize the posttranslocated elongation complex at a stall position. Therefore, at least two NTP substrates pair to DNA templated sites downstream of the active site. These results demonstrate the mechanisms of NTP loading and transcriptional efficiency and fidelity for human RNA polymerase II and indicate regulation of downstream bubble opening by NTPs.

Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation.

The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), in which the alpha1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the alpha1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in alpha1. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS. TFIIF 74(1-158) is defective because it fails to promote forward translocation. Deletion of the RAP74 alpha1 helix results in increased occupancy of the backtracking, cleavage, and restart pathways at a stall position, indicating reverse translocation of the elongation complex. During elongation, TFIIF 74(1-158) fails to support detectable nucleoside triphosphate (NTP)-driven translocation from a stall position and is notably defective in supporting bond completion (NTP-driven translocation coupled to pyrophosphate release) during the processive transition between bonds.

The highly conserved glutamic acid 791 of Rpb2 is involved in the binding of NTP and Mg(B) in the active center of human RNA polymerase II.

During transcription by RNA polymerase (RNAP) II, the incoming ribonucleoside triphosphate (NTP) enters the catalytic center in association with an Mg2+ ion, termed metal B [Mg(B)]. When bound to RNAP II, Mg(B) is coordinated by the β and γ phosphates of the NTP, Rpb1 residues D481 and D483 and Rpb2 residue D837. Rpb2 residue D837 is highly conserved across species. Notably, its neighboring residue, E836 (E791 in human RNAP II), is also highly conserved. To probe the role of E791 in transcription, we have affinity purified and characterized a human RNAP II mutant in which this residue was substituted for alanine. Our results indicate that the transcription activity of the Rpb2 E791A mutant is impaired at low NTP concentrations both in vitro and in vivo. They also revealed that both its NTP polymerization and transcript cleavage activities are decreased at low Mg concentrations. Because Rpb2 residue E791 appears to be located too far from the NTP–Mg(B) complex to make direct contact at either the entry (E) or addition (A) site, we propose alternative mechanisms by which this highly conserved residue participates in loading NTP–Mg(B) in the active site during transcription.

In vitro transcription termination activity of the Drosophila mitochondrial DNA-binding protein DmTTF

DmTTF is a Drosophila melanogaster mitochondrial DNA-binding protein which binds specifically to two homologous non-coding sequences located at the 3′ ends of blocks of genes encoded on opposite strands. In order to test whether this protein acts as transcription termination factor, we assayed the capacity of DmTTF to arrest in vitro the transcription catalyzed by mitochondrial and bacteriophage RNA polymerases. Experiments with human S-100 extracts showed that DmTTF is able to arrest the transcription catalyzed by human mitochondrial RNA polymerase bidirectionally, independently of the orientation of the protein–DNA complex. On the contrary when T3 or T7 RNA polymerases were used, we found that DmTTF prevalently arrests transcription when the DNA-binding site was placed in the reverse orientation with respect to the incoming enzymes. These results demonstrate that DmTTF is a transcription termination factor with a biased polarity and suggest that the DNA-bound protein is structurally asymmetrical, exposing two different faces to RNA polymerases travelling on opposite directions.

Cloning of short hairpin RNAs for gene knockdown in mammalian cells

. We and others have chosen to explore the shRNAs for several reasons: first, the considerable cost of chemically synthesized siRNAs; second, the possibility of enforceable and stable expression of shRNAs; and third, the availability of applications of expression constructs in primary cell types (for example, using retroviruses) and in whole organisms (for example, in mouse). We have developed a system to drive expression of shRNAs by placing them under the control of the human RNA polymerase III U6 small nuclear RNA (snRNA) promoter, which normally controls expression of small RNAs in cells. This system has now been demonstrated to be effective both in

Transcription Factors IIF and IIS and Nucleoside Triphosphate Substrates as Dynamic Probes of the Human RNA Polymerase II Mechanism

The mechanism for elongation catalyzed by human RNA polymerase II (RNAP II) has been analyzed using millisecond phase transient state kinetics. Here, we apply a running start, two-bond, double-quench protocol. Quenching the reaction with EDTA indicates NTP loading into the active site followed by rapid isomerization. HCl quenching defines the time of phosphodiester bond formation. Model-independent and global kinetic analyses were applied to simulate the RNAP II mechanism for forward elongation through the synthesis of two specific phosphodiester bonds, modeling rate data collected over a wide range of nucleoside triphosphate concentrations. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF alone and in the presence of TFIIF+TFIIS, providing detailed insight into the RNAP II mechanism and into processive RNA synthesis. RNAP II extends an RNA chain through a substrate induced-fit mechanism, termed NTP-driven translocation. After rapid isomerization, chemistry is delayed. At a stall point induced by withholding the next templated NTP, RNAP II fractionates into at least two active and one paused conformation, revealed as different forward rates of elongation. In the presence of TFIIF alone or in the presence of TFIIF+TFIIS, rapid rates are very similar; although, with TFIIF alone the complex is more highly poised for forward synthesis. Based on steady-state analysis, TFIIF was thought to suppress transcriptional pausing, but this view is misleading. TFIIF supports elongation and suppresses pausing by stabilizing the post-translocated elongation complex. When TFIIS is present, RNA cleavage and transcriptional restart pathways are supported, but TFIIS has a role in suppression of transient pausing, which is the most important contribution of TFIIS to elongation from a stall position.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Downstream DNA Selectively Affects a Paused Conformation of Human RNA Polymerase II

Transcriptional pausing by human RNA polymerase II (RNAPII) in the HIV-1 LTR is caused principally by a weak RNA:DNA hybrid that allows rearrangement of reactive or catalytic groups in the enzyme's active site. This rearrangement creates a transiently paused state called the unactivated intermediate that can backtrack into a more long-lived paused species. We report that three different regions of the not-yet-transcribed DNA just downstream of the pause site affect the duration of the HIV-1 pause, and also can influence pause formation. Downstream DNA in at least one region, a T-tract from +5 to +8, increases pause duration by specifically affecting the unactivated intermediate, without corresponding effects on the active or backtracked states. We suggest this effect depends on RNAPII-modulated DNA plasticity and speculate it is mediated by the “trigger loop” thought to participate in RNAP's catalytic cycle. These findings provide a new framework for understanding downstream DNA effects on RNAP.

Transcription Factor (TF)-like Nuclear Regulator, the 250-kDa Form of Homo sapiens TFIIIB″, Is an Essential Component of Human TFIIIC1 Activity

The general human RNA polymerase III transcription factor (TF) IIIC1 has hitherto been ill defined with respect to the polypeptides required for reconstitution of its activity. Here we identify Homo sapiens TFIIIB" (HsBdp1) as an essential component of hTFIIIC1 and hTFIIIC1-like activities. Several forms of HsBdp1 are described. The 250-kDa form of HsBdp1, also designated the "transcription factor-like nuclear regulator," strictly co-eluted with TFIIIC1 activity over multiple chromatographic purification steps as revealed by Western blot with anti-HsBdp1 antibodies and by MALDI-TOF analysis. In addition, TFIIIC1 activity could be depleted from partially purified fractions with anti-HsBdp1 antibodies but not with control antibodies. Moreover, highly purified recombinant HsBdp1 could replace TFIIIC1 activity in reconstituted transcription of the VAI gene in vitro. Furthermore, smaller proteins of approximately 90-150 kDa that were recognized by anti-HsBdp1 antibodies co-eluted with TFIIIC1-like activity. Finally, cytoplasmic extracts from differentiated mouse F9 fibroblast cells that lacked TFIIIC1 activity could be made competent for transcription of the VA1 gene by the addition of TFIIIC1, TFIIIC1-like, or recombinant HsBdp1. These results suggest that HsBdp1 proteins represent essential components of TFIIIC1 and TFIIIC1-like activities.