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RNA Polymerase II Elongation at the Crossroads of Transcription and Alternative Splicing  [PDF]
Manuel de la Mata,Manuel J. Mu?oz,Mariano Alló,Juan Pablo Fededa,Ignacio E. Schor,Alberto R. Kornblihtt
Genetics Research International , 2011, DOI: 10.4061/2011/309865
Abstract: The elongation phase of transcription lies at the core of several simultaneous and coupled events leading to alternative splicing regulation. Although underestimated in the past, it is at this phase of the transcription cycle where complexes affecting the transcription machinery itself, chromatin structure, posttranscriptional gene regulation and pre-mRNA processing converge to regulate each other or simply to consolidate higher-order complexes and functions. This paper focuses on the multiple processes that take place during transcription elongation which ultimately regulate the outcome of alternative splicing decisions. 1. Introduction Regulation of gene expression was originally conceived as a hierarchy of steps linked together on a time scale and physically separated in different cell compartments in accordance with the central dogma of biology. This concept has long been abandoned, with a significant accumulation of evidence describing an extensive network of events, encompassing transcription, mRNA processing, chromatin regulation, and posttranscriptional gene regulation, which take place simultaneously and in a mutually regulated or coupled manner [1, 2]. Distinctions between complexes and processes governing gene expression have been blurred to a large extent, adding complexity to the ever-increasing fraction of genes subjected to alternative promoter usage, alternative splicing (AS) (>90% genes), alternative polyadenylation, editing, and posttranscriptional gene silencing by small RNAs [3, 4]. Additionally, this complexity takes a new dimension when studied in the context of chromatin and its regulation upon gene expression (for reviews, see [5–7]). This paper will focus on the main features of coupling between transcriptions elongation and splicing, and its implications on AS regulation. 2. The Benefits of Coupling Initial visualization of Drosophila-embryo nascent transcripts by electron microscopy, showed that splicing can occur cotranscriptionally [8]. This was later directly demonstrated for the human dystrophin gene [9], which spans 2400?kb and can take 16?hr to complete transcription. More recently, a quantitative study of the c-Src and fibronectin mRNAs compared chromatin-bound and nucleoplasmic RNA fractions. There, it was shown that most introns are excised efficiently in the chromatin-bound fractions, with a gradient of cotranscriptional splicing efficiency decreasing from promoter-proximal to promoter-distal introns, that is, the direction of transcription [10]. One implication of the cotranscriptional nature of splicing is that the
Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation  [PDF]
Pedro Rafael Costa, Marcio Luis Acencio, Ney Lemke
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057328
Abstract: The transcription process is crucial to life and the enzyme RNA polymerase (RNAP) is the major component of the transcription machinery. The development of single-molecule techniques, such as magnetic and optical tweezers, atomic-force microscopy and single-molecule fluorescence, increased our understanding of the transcription process and complements traditional biochemical studies. Based on these studies, theoretical models have been proposed to explain and predict the kinetics of the RNAP during the polymerization, highlighting the results achieved by models based on the thermodynamic stability of the transcription elongation complex. However, experiments showed that if more than one RNAP initiates from the same promoter, the transcription behavior slightly changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAPs and predicts their cooperative behavior during multi-round transcription generalizing the Bai et al. stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica? and compared the results of the single and the multiple-molecule transcription with experimental results and other theoretical models. Our multi-round approach can recover several expected behaviors, showing that the transcription process for the studied sequences can be accelerated up to 48% when collisions are allowed: the dwell times on pause sites are reduced as well as the distance that the RNAPs backtracked from backtracking sites.
Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes  [PDF]
Lu Zhang?,Daniel-Adriano Silva?,Fátima Pardo-Avila?,Dong Wang?,Xuhui Huang
PLOS Computational Biology , 2015, DOI: 10.1371/journal.pcbi.1004354
Abstract: The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the “secondary channel” is the only route for NTP to reach the active site of the enzyme or if the “main channel” could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.
Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain  [PDF]
Jeetendra Kumar Nag,Nidhi Shrivastava,Dhanvantri Chahar,Chhedi Lal Gupta,Preeti Bajpai,Shailja Misra-Bhattacharya
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002930
Abstract: Objectives Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains. Methodology/Principal Findings Biophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ββ′σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ββ′σ subunits of polymerase. Conclusions/Significance To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of this α-proteobacteria and in deciphering the role of Wol GreA in filarial development.
A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation  [PDF]
Lin-Tai Da?,Chao E?,Baogen Duan?,Chuanbiao Zhang?,Xin Zhou?,Jin Yu
PLOS Computational Biology , 2015, DOI: 10.1371/journal.pcbi.1004624
Abstract: Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase.
Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective  [PDF]
Kris Brannan,David L. Bentley
Genetics Research International , 2012, DOI: 10.1155/2012/170173
Abstract: The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5′ ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression. 1. Introduction The initiation phase of the RNA polymerase II (pol II) transcription cycle involves multiple events, including recruitment of general transcription factors and pol II to the promoter, melting of the DNA template, initiation of RNA synthesis, and pol II promoter clearance, which marks entry into the elongation phase. The stochastic nature of all of these steps poses a potential problem if it becomes necessary to mount a rapid activation of transcription. Following initiation pol II often encounters a rate-limiting barrier that appears to lie between early elongation and productive elongation. The transition between these two phases of the transcription cycle has now been characterized as a powerful regulatory switch used to increase or decrease gene expression in a signal-responsive fashion. Here we review the early discoveries that laid the foundation for a detailed understanding of transcriptional regulation at this transition. 2. Early Evidence of Polymerase Pausing and Premature Termination in DNA Viruses Nearly 30 years ago it was reported by the late Yosef Aloni and colleagues that run-on transcripts made in nuclei from SV40 infected cells were strongly biased toward the 5′ end of the late transcription unit suggesting that pol II accumulated in the promoter-proximal region [1]. Analysis of labeled RNA extended on viral transcription complexes (VTCs) assembled in vivo and purified from infected cells revealed two additional unusual features of transcription from the late promoter. First, two pause sites were mapped around positions +15 and +40 relative to the start site by identifying the junctions between unlabelled RNA made in vivo and labeled RNA extended in vitro [2]. Second, a major product of transcription on VTCs is a discrete 93–95 base RNA, that is, prematurely terminated near a potential hairpin loop structure. Similar
Control of eukaryotic transcription elongation
Fred Winston
Genome Biology , 2001, DOI: 10.1186/gb-2001-2-2-reviews1006
Abstract: It has been clear for over 20 years that the control of transcription elongation is a prominent mechanism of gene regulation in prokaryotes. In eukaryotes, although this level of regulation was clearly demonstrated in 1988 [1], it is mainly over the past five years that transcription elongation has blossomed into a broadly active area of investigation [2,3]. For example, several recent studies of the human immunodeficiency virus (HIV) Tat protein, a transcriptional activator that stimulates transcription past a pause site, have helped us to understand factors required for elongation [4,5].Three transcription-elongation factors that have emerged as playing critical roles are the Spt4, Spt5, and Spt6 proteins. These three proteins, conserved among eukaryotes, were initially discovered by genetic studies in Saccharomyces cerevisiae [6]. Further studies in S. cerevisiae suggested that Spt4, Spt5, and Spt6 are essential for transcription via the modulation of chromatin structure [7,8]. Spt4 and Spt5 are tightly associated in a complex, while the physical association of the Spt4-Spt5 complex with Spt6 appears to be considerably weaker [9,10]. Evidence over the past two years has demonstrated that Spt4, Spt5, and Spt6 play roles in transcription elongation in both yeast and humans [9,10], including a role in activation by Tat [11]. The breadth of the requirement for these factors in multicellular eukaryotes was not known, however. Now, four papers [12,13,14,15] have illuminated the roles of these factors in vivo. These new studies, taken together with earlier work, portray Spt4, Spt5, and Spt6 as general transcription-elongation factors, controlling transcription both positively and negatively in important regulatory and developmental roles.Two recent studies [12,13] of Spt5 and Spt6 have provided the first view of their roles in vivo in a multicellular eukaryote, in this case Drosophila melanogaster. In both studies, specific antisera were used to determine the localizati
Accuracy of the serological ELISA test compared with the polymerase chain reaction for the diagnosis of cytomegalovirus infection in pregnancy
Parmigiani, Silvana Varella;Barini, Ricardo;Costa, Sandra Cecília Botelho;Amaral, Eliana;Silva, José Carlos Gama da;Pinto e Silva, Jo?o Luiz de Carvalho;
Sao Paulo Medical Journal , 2003, DOI: 10.1590/S1516-31802003000300002
Abstract: context: the most frequently used methods for detecting antibodies are the indirect immunofluorescence test and the enzymatic immunoassay (elisa). the polymerase chain reaction is a molecular biology technique in which the production of large amounts of specific dna fragments is induced from very low concentrations of complex substrates aloowing the detection of very low amounts of viral particles. objective: to assess the accuracy of serological/elisa tests in comparison with the polymerase chain reaction in maternal blood to diagnose cytomegalovirus infection. design: a descriptive study was performed. setting: high-risk outpatient clinic of campinas university (unicamp). participants: we selected 243 pregnant women. all of them had been indicated for blood sampling because of suspicions of cytomegalovirus infection and also because of other infections. main measurements: the group was tested for cytomegalovirus. serological tests were run and compared to the polymerase chain reaction, which was considered to be the gold standard. status analyses were done using fisher's exact test, via the sas software. results: the previous cytomegalovirus infection rate was 94.6%. the main reasons for inclusion in the study were fetal nervous system malformation (25.5%), maternal toxoplasmosis (25.5%) and rh isoimmunization (14.8%). only two women were included because of positive serological immunoglobulin m test for cytomegalovirus. the sensitivity and specificity of the serological tests were 94% and 6% for immunoglobulin g. conclusion: serological tests had lower sensitivity in comparison with the polymerase chain reaction test when diagnosing cytomegalovirus infection. the consequences of positive polymerase chain reaction and negative immunoglobulin m in women remain unknown.
The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency  [PDF]
Kyle A. Nilson,David H. Price
Genetics Research International , 2011, DOI: 10.4061/2011/726901
Abstract: HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR) is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II), leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure. 1. Introduction According to the 2010 UNAIDS AIDS Epidemic Update, over 33 million people live with human immunodeficiency virus (HIV) type 1, a number that is increasing due to a combination of improved treatment and continued transmission. Upon crossing the mucosa, HIV docks with CD4+ cells such as T-lymphocytes and macrophages, fuses with the host cell, and releases viral single-stranded RNA, reverse transcriptase, and integrase into the cytoplasm. Reverse transcriptase converts the HIV RNA into double-stranded DNA, at which point integrase chaperones the viral DNA into the nucleus for integration into the host genome. An initial round of host-induced gene expression by Pol II results in expression of Tat, the primary transactivator of HIV, which then recruits the positive transcription elongation factor P-TEFb containing Cdk9 and Cyclin T1 to the HIV LTR [1, 2]. This leads to increased viral gene expression and, eventually, replication of the HIV genome, assembly into new viral particles, and budding. HIV is capable of establishing life-long latent infection by suppressing its transcription, thus evading current antiretroviral therapies [3]. How HIV subverts Pol II elongation control during both active and latent infections has received a significant amount of attention, and it is hoped that these inquires will lead to the development of more effective treatments and an eventual cure. Regulation of transcription of many human genes is accomplished by a process termed RNA polymerase II elongation control, and, after integration, the HIV LTR falls under this control. In fact, the HIV LTR has been used as a model to study the regulation of transcription at the level of elongation. In general, most
Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir
Rachel B Gill, Samuel L Frederick, Caroll B Hartline, Sunwen Chou, Mark N Prichard
Virology Journal , 2009, DOI: 10.1186/1743-422x-6-9
Abstract: Human cytomegalovirus (HCMV) is a ubiquitous virus that can be problematic in immunocompromised populations, including individuals with AIDS or recipients of allograft transplants. It is the most common congenital infection in the United States [1] and sequela include permanent neurological deficits, including hearing loss [2,3]. Ganciclovir (GCV), foscarnet and cidofovir (CDV) have all been approved for the treatment of HCMV infection, but each drug is associated with dose-limiting toxicities [4]. The benzimidazole L-riboside maribavir (MBV) is currently in Phase III clinical trials for the treatment of HCMV infections and inhibits viral replication by a distinct mechanism involving the direct inhibition of UL97 kinase activity [4-7]. While this drug clearly inhibits the enzymatic activity of the UL97 kinase in infected cells, the consequences of its inhibition are complex and incompletely understood as the kinase affects many cellular and viral processes.The UL97 serine/threonine kinase is expressed early in infection and is found within the tegument of infectious virions [8,9]. Although the kinase is not required for viral replication, null mutants exhibit severe replication deficits [10], which is consistent with the inhibitory effects of MBV [5]. This enzyme has been shown to phosphorylate viral proteins including itself, ppUL44 and pp65 [11-13], as well as the large subunit of RNA polymerase II, eukaryotic elongation factor 1delta, P32 and lamins A/C [14-16]. The tumor suppressor retinoblastoma (Rb) has also been shown to be hyperphosphorylated in cells infected with HCMV [17], and this phosphorylation is dependent on UL97 kinase activity [18]. This report also showed that mutations in either the essential lysine (K355) or the conserved LxCxE Rb-binding motif in the amino terminus of pUL97 reduced the inactivation of Rb [18]. A separate study showed that the kinase phosphorylated Rb directly and did not require other proteins [19]. This activity is intriguing
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