Elongation Phase Of Transcription Research Articles

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

To Splice or to Transcribe: SKIP-Mediated Environmental Fitness and Development in Plants

Gene expression in eukaryotes is controlled at multiple levels, including transcriptional and post-transcriptional levels. The transcriptional regulation of gene expression is complex and includes the regulation of the initiation and elongation phases of transcription. Meanwhile, the post-transcriptional regulation of gene expression includes precursor messenger RNA (pre-mRNA) splicing, 5′ capping, and 3′ polyadenylation. Among these events, pre-mRNA splicing, conducted by the spliceosome, plays a key role in the regulation of gene expression, and the efficiency and precision of pre-mRNA splicing are critical for gene function. Ski-interacting protein (SKIP) is an evolutionarily conserved protein from yeast to humans. In plants, SKIP is a bifunctional regulator that works as a splicing factor as part of the spliceosome and as a transcriptional regulator via interactions with the transcriptional regulatory complex. Here, we review how the functions of SKIP as a splicing factor and a transcriptional regulator affect environmental fitness and development in plants.

De novo MEPCE nonsense variant associated with a neurodevelopmental disorder causes disintegration of 7SK snRNP and enhanced RNA polymerase II activation

In eukaryotes, the elongation phase of transcription by RNA polymerase II (RNAP II) is regulated by the transcription elongation factor b (P-TEFb), composed of Cyclin-T1 and cyclin-dependent kinase 9. The release of RNAP II is mediated by phosphorylation through P-TEFb that in turn is under control by the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. The 7SK snRNP consists of the 7SK non-coding RNA and the proteins MEPCE, LARP7, and HEXIM1/2. Biallelic LARP7 loss-of-function variants underlie Alazami syndrome characterized by growth retardation and intellectual disability. We report a boy with global developmental delay and seizures carrying the de novo MEPCE nonsense variant c.1552 C > T/p.(Arg518*). mRNA and protein analyses identified nonsense-mediated mRNA decay to underlie the decreased amount of MEPCE in patient fibroblasts followed by LARP7 and 7SK snRNA downregulation and HEXIM1 upregulation. Reduced binding of HEXIM1 to Cyclin-T1, hyperphosphorylation of the RNAP II C-terminal domain, and upregulated expression of ID2, ID3, MRPL11 and snRNAs U1, U2 and U4 in patient cells are suggestive of enhanced activation of P-TEFb. Flavopiridol treatment and ectopic MEPCE protein expression in patient fibroblasts rescued increased expression of six RNAP II-sensitive genes and suggested a possible repressive effect of MEPCE on P-TEFb-dependent transcription of specific genes.

The cutting edge of archaeal transcription.

The archaeal RNA polymerase (RNAP) is a double-psi β-barrel enzyme closely related to eukaryotic RNAPII in terms of subunit composition and architecture, promoter elements and basal transcription factors required for the initiation and elongation phase of transcription. Understanding archaeal transcription is, therefore, key to delineate the universally conserved fundamental mechanisms of transcription as well as the evolution of the archaeo-eukaryotic transcription machineries. The dynamic interplay between RNAP subunits, transcription factors and nucleic acids dictates the activity of RNAP and ultimately gene expression. This review focusses on recent progress in our understanding of (i) the structure, function and molecular mechanisms of known and less characterized factors including Elf1 (Elongation factor 1), NusA (N-utilization substance A), TFS4, RIP and Eta, and (ii) their evolution and phylogenetic distribution across the expanding tree of Archaea.

A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans.

The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation.

Mechanisms of HIV Transcriptional Regulation by Drugs of Abuse.

There is a strong correlation between the use and abuse of illicit drugs and the spread of Human Immunodeficiency Virus (HIV). It is well established that illicit drugs users are a high risk population for infection with HIV with an increased rate of HIV transmission and replication. Cocaine, amphetamine, methamphetamine, heroin and morphine stand out as the most frequently abused illicit drugs and their use correlates well with HIV infection and AIDS progression. Notably, the high incidence of HIV infection in illicit drug abusers is primarily due to high risk activities such as needle sharing and unprotected sex. Several studies have also demonstrated that drugs of abuse increase viral RNA concentrations by enhancing HIV replication, in particular in the central nervous system (CNS). The CNS is a common target for both drugs of abuse and HIV, and their synergistic action accelerates neuronal injury and cognitive impairment. In order to generate complete genomic transcripts, HIV gene expression has to progress through both the initiation and elongation phases of transcription, which requires coordinated action of different transcription factors. In this review, we will provide the latest updates of the molecular mechanisms that regulate HIV transcription and discuss how drugs of abuse, such as cocaine, amphetamine, methamphetamine, heroin and morphine, modulate those mechanisms to upregulate HIV transcription and eventually HIV replication.

Structural basis for recognition of histone H3K36me3 nucleosome by human de novo DNA methyltransferases 3A and 3B.

DNA methylation is an important epigenetic modification involved in chromatin organization and gene expression. The function of DNA methylation depends on cell context and is correlated with histone modification patterns. In particular, trimethylation of Lys36 on histone H3 tail (H3K36me3) is associated with DNA methylation and elongation phase of transcription. PWWP domains of the de novo DNA methyltransferases DNMT3A and DNMT3B read this epigenetic mark to guide DNA methylation. Here we report the first crystal structure of the DNMT3B PWWP domain-H3K36me3 complex. Based on this structure, we propose a model of the DNMT3A PWWP domain-H3K36me3 complex and build a model of DNMT3A (PWWP-ADD-CD) in a nucleosomal context. The trimethylated side chain of Lys36 (H3K36me3) is inserted into an aromatic cage similar to the "Royal" superfamily domains known to bind methylated histones. A key interaction between trimethylated Lys36 and a conserved water molecule stabilized by Ser270 explains the lack of affinity of mutated DNMT3B (S270P) for the H3K36me3 epigenetic mark in the ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome. The model of the DNMT3A-DNMT3L heterotetramer in complex with a dinucleosome highlights the mechanism for recognition of nucleosome by DNMT3s and explains the periodicity of de novo DNA methylation.

The effects of cocaine on HIV transcription.

Illicit drug users are a high-risk population for infection with the human immunodeficiency virus (HIV). A strong correlation exists between prohibited drug use and an increased rate of HIV transmission. Cocaine stands out as one of the most frequently abused illicit drugs, and its use is correlated with HIV infection and disease progression. The central nervous system (CNS) is a common target for both drugs of abuse and HIV, and cocaine intake further accelerates neuronal injury in HIV patients. Although the high incidence of HIV infection in illicit drug abusers is primarily due to high-risk activities such as needle sharing and unprotected sex, several studies have demonstrated that cocaine enhances the rate of HIV gene expression and replication by activating various signal transduction pathways and downstream transcription factors. In order to generate mature HIV genomic transcript, HIV gene expression has to pass through both the initiation and elongation phases of transcription, which requires discrete transcription factors. In this review, we will provide a detailed analysis of the molecular mechanisms that regulate HIV transcription and discuss how cocaine modulates those mechanisms to upregulate HIV transcription and eventually HIV replication.

Transcriptional properties of mammalian Elongin A and its role in stress response (944.1)

Transcriptional elongation factor Elongin A exhibits an activity to repress the temporary pause of RNA polymerase II (RNAPII) on the DNA template. The role of Elongin A in RNAPII transcription in mammalian cells has not, however, been clearly established. In this study, we investigate the function of Elongin A in RNAPII transcription. First, we examined immunoprecipitation and immunostaining. These showed that Elongin A binds to the phosphorylated form of RNAPII (IIO) at both Ser5 and Ser2 and partially colocalized with the RNAPIIO and newly transcribed RNA. Significantly, Western blotting and qRT‐PCR showed that Elongin A was required for the maximal induction of stress response gene ATF3 and p21. ChIP assay showed that Elongin A occupancy was increased on ATF3, p21, c‐Myc and c‐jun genes after doxorubicin treatment. Furthermore, we performed re‐introduction assay of various mutants of Elongin A. It indicated that expressions of ATF3 and p21 gene were dependent on elongation activity of Elongin A. By contrast, polyubiquitination of RNAPII was dependent on ubiqutination activity of Elongin A. Taken together, our data provide the evidence that mammalian Elongin A is involved in the elongation phase of transcription in vivo and plays a crucial role in response to stress stimuli. Furthermore, our data indicated that two activities of Elongin A are independent of one another in response to stress.

Single Molecule Investigation of RNA Polymerase I using Multiplexed Tethered Particle Motion

In eukaryotic cells, RNA Polymerase I (Pol I), which produces ribosomal RNA (rRNA), catalyzes up to 80% of all transcriptional activity in growing cells, and its regulation is an emerging field of study for anti-cancer drug development. In order to gather more comprehensive information about ribosomal RNA production by Pol I, we conducted single molecule experiments to characterize rates and pauses in the elongation phase of transcription by wild-type and mutant Pol I. Transcription by individual molecules was based on real-time measurements using custom multiplexed Tethered Particle Motion (TPM) microscopy. Microspheres were attached to one end of a DNA template engaged in an elongation complex with a Pol I molecule immmuno-imobilized on a coverslip. The range of Brownian motion of the microsphere was a function of the tether length that in turn depended on the progress of the polymerase. Either the lack of a full complement of associated factors or the constitution of in vitro transcription assay limited of polymerase activity to only 1-2%. In order to increase the throughput of the transcription assay, we increased the field of view and implemented a particle-tracking algorithm to track a high number of beads simultaneously in real time. The time courses of rare transcription events were analyzed for velocity and pausing. The processivity of Pol I without tension on the DNA template was remarkably lower than observed in studies of Pol II transcribing DNA templates under a few picoNewtons of tension. Furthermore, as reported previously based on bulk experiments, the average elongation rate of an F1205H mutant was lower than that of wild-type Pol I. At least six possible pause sites were also identified including one recently found in bulk transcription study.

The RNA polymerase trigger loop functions in all three phases of the transcription cycle

The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA–RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2’-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites.

Effects of Viscogens on RNA Transcription inside Reovirus Particles

The dsRNA genome of mammalian reovirus (MRV), like the dsDNA genomes of herpesviruses and many bacteriophages, is packed inside its icosahedral capsid in liquid-crystalline form, with concentrations near or more than 400 mg/ml. Viscosity in such environments must be high, but the relevance of viscosity for the macromolecular processes occurring there remains poorly characterized. Here, we describe the use of simple viscogens, glycerol and sucrose, to examine their effects on RNA transcription inside MRV core particles. Transcription inside MRV cores was strongly inhibited by these agents and to a greater extent than either predicted by theory or exhibited by a nonencapsidated transcriptase, suggesting that RNA transcription inside MRV cores is unusually sensitive to viscogen effects. The elongation phase of transcription was found to be a primary target of this inhibition. Similar results were obtained with particles of a second dsRNA virus, rhesus rotavirus, from a divergent taxonomic subfamily. Polymeric viscogens such as polyethylene glycol also inhibited RNA transcription inside MRV cores, but in a size-limited manner, suggesting that diffusion through channels in the MRV core is required for their activity. Modeling of the data suggested that the inherent intracapsid viscosity of both reo- and rotavirus is indeed high, two to three times the viscosity of water. The capacity for quantitative comparisons of intracapsid viscosity and effects of viscogens on macromolecular processes in confined spaces should be similarly informative in other systems.

Cycling through transcription with the RNA polymerase F/E (RPB4/7) complex: structure, function and evolution of archaeal RNA polymerase

RNA polymerases (RNAPs) from the three domains of life, Bacteria, Archaea and Eukarya, are evolutionarily related and thus have common structural and functional features. Despite the radically different morphology of Archaea and Eukarya, their RNAP subunit composition and utilisation of basal transcription factors are almost identical. This review focuses on the multiple functions of the most prominent feature that differentiates these enzymes from the bacterial RNAP – a stalk-like protrusion, which consists of the heterodimeric F/E subcomplex. F/E is highly versatile, it facilitates DNA strand-separation during transcription initiation, increases processivity during the elongation phase of transcription and ensures efficient transcription termination.

Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription

Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defense against parasitic nucleic acids, and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei 1-2. For instance, in plants and fungi siRNAs associate with nascent transcripts and direct chromatin and/or DNA modifications 1-2. To further understand the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in C. elegans nuclei 3. Here we show that nrde-2 encodes an evolutionarily conserved protein that is required for small interfering (si)RNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct a NRDE-2-dependent silencing of pre-mRNAs 3’ to sites of RNAi, a NRDE-2-dependent accumulation of RNA Polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3’ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation; siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.

Molecular basis of histone H3K36me3 recognition by the PWWP domain of Brpf1

Trimethylation of Lys36 in histone H3 (H3K36me3) coordinates events associated with the elongation phase of transcription and is also emerging as an important epigenetic regulator of cell growth and differentiation. We have identified the PWWP domain of bromo and plant homeodomain (PHD) finger-containing protein 1 (BRPF1) as a H3K36me3 binding module and have determined the structure of this domain in complex with an H3K36me3-derived peptide.

Dynamic bookmarking of primary response genes by p300 and RNA polymerase II complexes

Profiling the dynamic interaction of p300 with proximal promoters of human T cells identified a class of genes that rapidly coassemble p300 and RNA polymerase II (pol II) following mitogen stimulation. Several of these p300 targets are immediate early genes, including FOS, implicating a prominent role for p300 in the control of primary genetic responses. The recruitment of p300 and pol II rapidly transitions to the assembly of several elongation factors, including the positive transcriptional elongation factor (P-TEFb), the bromodomain-containing protein (BRD4), and the elongin-like eleven nineteen lysine-rich leukemia protein (ELL). However, transcription at many of these rapidly induced genes is transient, wherein swift departure of P-TEFb, BRD4, and ELL coincides with termination of transcriptional elongation. Unexpectedly, both p300 and pol II remain accumulated or "bookmarked" at the proximal promoter long after transcription has terminated, demarking a clear mechanistic separation between the recruitment and elongation phases of transcription in vivo. The bookmarked pol II is depleted of both serine-2 and serine-5 phosphorylation of its C-terminal domain and remains proximally positioned at the promoter for hours. Surprisingly, these p300/pol II bookmarked genes can be readily reactivated, and elongation factors can be reassembled by subsequent addition of nonmitogenic agents that, alone, have minimal effects on transcription in the absence of prior preconditioning by mitogen stimulation. These findings suggest that p300 is likely to play an important role in biological processes in which transcriptional bookmarking or preconditioning influences cellular growth and development through the dynamic priming of genes for response to rechallenge by secondary stimuli.

Isolation and functional analysis of RNA polymerase II elongation complexes

The elongation phase of transcription by RNA polymerase II (RNAP II) is tightly controlled by a large number of transcription elongation factors. Here we describe experimental approaches for the isolation of RNAPII elongation complexes in vitro and the use of these complexes in the examination of the function of a variety of factors. The methods start with formation of elongation complexes on DNA templates immobilized to paramagnetic beads. Elongation is halted by removing the nucleotides and the ternary elongation complexes are then stripped of factors by a high salt wash. The effect of any factor or mixture of factors on elongation is determined by adding the factor(s) along with nucleotides and observing the change in the pattern of RNAs generated. Association of a factor with elongation complexes can be examined using an elongation complex-electrophoretic mobility shift assay (EC-EMSA) in which elongation complexes that have been liberated from the beads are analyzed on a native gel. Besides being used to dissect the mechanisms of elongation control, these experimental systems are useful for analyzing the function of termination factors and mRNA processing factors. Together these experimental systems permit detailed characterization of the molecular mechanisms of elongation, termination, and mRNA processing factors by providing information concerning both physical interactions with and functional consequences of the factors on RNAPII elongation complexes.

The Structure of a Transcribing T7 RNA Polymerase in Transition from Initiation to Elongation

Structural studies of the T7 bacteriophage DNA-dependent RNA polymerase (T7 RNAP) have shown that the conformation of the amino-terminal domain changes substantially between the initiation and elongation phases of transcription, but how this transition is achieved remains unclear. We report crystal structures of T7 RNAP bound to promoter DNA containing either a 7- or an 8-nucleotide (nt) RNA transcript that illuminate intermediate states along the transition pathway. The amino-terminal domain comprises the C-helix subdomain and the promoter binding domain (PBD), which consists of two segments separated by subdomain H. The structures of the intermediate complex reveal that the PBD and the bound promoter rotate by approximately 45 degrees upon synthesis of an 8-nt RNA transcript. This allows the promoter contacts to be maintained while the active site is expanded to accommodate a growing heteroduplex. The C-helix subdomain moves modestly toward its elongation conformation, whereas subdomain H remains in its initiation- rather than its elongation-phase location, more than 70 angstroms away.

RNA Polymerase Elongation Factors

The elongation phase of transcription by RNA polymerase is highly regulated and modulated. Both general and operon-specific elongation factors determine the local rate and extent of transcription to coordinate the appearance of transcript with its use as a messenger or functional ribonucleoprotein or regulatory element, as well as to provide operon-specific gene regulation.

Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay.

The elongation phase of transcription by RNA polymerase II (RNAP II) is controlled by a carefully orchestrated series of interactions with both negative and positive factors. However, due to the limitations of current methods and techniques, not much is known about whether and how these proteins physically associate with the engaged polymerases. To gain insight into the detailed mechanisms involved, we established an experimental system for analyzing direct factor interactions to RNAP II elongation complexes on native gels, namely elongation complex electrophoretic mobility shift assay (EC-EMSA). This new assay effectively allowed detection of interactions of TFIIF, TTF2, TFIIS, DSIF and P-TEFb with elongation complexes generated from a natural promoter using an immobilized template. As an application of this assay system, we characterized the association of transcription elongation factor DSIF with RNAP II elongation complexes and discovered that the nascent transcript facilitated recruitment of DSIF. Examples of how the system can be manipulated to address different questions are provided. EC-EMSA should be useful for further investigation of factor interactions with RNAP II elongation complexes.