Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation

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. © 2013 Costa et al.

Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation.

Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.

Protein-RNA interactions in the active center of transcription elongation complex.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.

Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex.

The human immunodeficiency virus type 1 transactivator protein, Tat, stimulates transcriptional elongation from the viral long terminal repeat. To test whether Tat associates directly with activated transcription complexes, we have used the lac repressor protein (LacR) to "trap" elongating RNA polymerases. The arrested transcription complexes were purified by binding biotinylated templates to streptaviridin-coated magnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II. The Tat protein copurified with RNA polymerase bound to wild-type templates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response element (TAR) RNA. We conclude that Tat and cellular cofactors become attached to the transcription complex during its transit through TAR.

Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.

RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Regulation of c-fos expression by RNA polymerase elongation competence.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Analyse de la localisation génomique et identification de nouvelles fonctions des sous-unités Rpb4/Rpb7 de l’ARN polymérase II et des facteurs TFIIF, TFIIS et UBR5

Grâce à un grand nombre d’études biochimiques, génétiques et structurales effectuées dans les dernières années, des avancements considérables ont été réalisés et une nouvelle vision du processus par lequel la machinerie transcriptionnelle de l’ARN polymérase II (Pol II) décode l’information génétique a émergé. De nouveaux indices ont été apportés sur la diversité des mécanismes de régulation de la transcription, ainsi que sur le rôle des facteurs généraux de transcription (GTFs) dans cette diversification. Les travaux présentés dans cette thèse amènent de nouvelles connaissances sur le rôle des GTFs humains dans la régulation des différentes étapes de la transcription. Dans la première partie de la thèse, nous avons analysé la fonction de la Pol II et des GTFs humains, en examinant de façon systématique leur localisation génomique. Les patrons obtenus par immunoprécipitation de la chromatine (ChIP) des versions de GTFs portant une étiquette TAP (Tandem-Affinity Purification) indiquent de nouvelles fonctions in vivo pour certains composants de cette machinerie et pour des éléments structuraux de la Pol II. Nos résultats suggèrent que TFIIF et l’hétérodimère Rpb4–Rpb7 ont une fonction spécifique pendant l’étape d’élongation transcriptionnelle in vivo. De plus, notre étude amène une première image globale de la fonction des GTFs pendant la réaction transcriptionnelle dans des cellules mammifères vivantes. Deuxièmement, nous avons identifié une nouvelle fonction de TFIIS dans la régulation de CDK9, la sous-unité kinase du facteur P-TEFb (Positive Transcription Elongation Factor b). Nous avons identifié deux nouveaux partenaires d’interaction pour TFIIS, soit CDK9 et la E3 ubiquitine ligase UBR5. Nous montrons que UBR5 catalyse l’ubiquitination de CDK9 in vitro. De plus, la polyubiquitination de CDK9 dans des cellules humaines est dépendante de UBR5 et TFIIS. Nous montrons aussi que UBR5, CDK9 and TFIIS co-localisent le long du gène  fibrinogen (FBG) et que la surexpression de TFIIS augmente les niveaux d’occupation par CDK9 de régions spécifiques de ce gène, de façon dépendante de UBR5. Nous proposons que TFIIS a une nouvelle fonction dans la transition entre les étapes d’initiation et d’élongation transcriptionnelle, en régulant la stabilité des complexes CDK9-Pol II pendant les étapes précoces de la transcription.

Cockayne syndrome group B protein enhances elongation by RNA polymerase II

Cockayne syndrome (CS) is characterized by impaired physical and mental development. Two complementation groups, CSA and CSB, have been identified. Here we report that the CSB gene product enhances elongation by RNA polymerase II. CSB stimulated the rate of elongation on an undamaged template by a factor of about 3. A thymine-thymine cyclobutane dimer located in the template strand is known to be a strong block to transcription. Addition of CSB to the blocked polymerase resulted in addition of one nucleotide to the nascent transcript. Finally, addition of transcription factor IIS is known to cause polymerase blocked at a thymine-thymine cyclobutane dimer to digest its nascent transcript, and CSB counteracted this transcript shortening action of transcription factor IIS. Thus a deficiency in transcription elongation may contribute to the CS phenotype.

A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Transcription in archaea: similarity to that in eucarya.

We present homologies between archaeal and eucaryal DNA-dependent RNA polymerase (RNAP) subunits and transcription factors. The sequences of the Sulfolobus acidocaldarius subunits D, E, and N and alignments with eucaryal homologs are presented here. The similarities between archaeal transcription factors and their eucaryal homologs TFIIB and TBP have been established in other laboratories. The archaeal RNAP subunits H, K, and N, respectively, show high sequence similarity to ABC27, ABC23, and ABC10 beta (found in all three eucaryal RNAPs); subunit D, to AC40 (common to polymerase II and polymerase III) and B44 (polymerase II); and subunit L, to AC19 and B12.5. The similarity of subunit D and its eucaryal homologs to bacterial alpha is limited to the "alpha-motif," which is also present in subunit L and its eucaryal homologs. Genes encoding homologs of the related eucaryal RNAP subunits A12.2/B12.6 and also homologs of eucaryal transcription elongation factors of the TFIIS family have been detected in Sulfolobus acidocaldarius and Thermococcus celer. In archaea, the protein is not an RNAP subunit. Together with the sequence similarities between archaeal box A-containing and eucaryal TATA box-containing promoters, this shows that the archaeal and eucaryal transcription systems are truly homologous and that they differ structurally and functionally from the bacterial transcription machinery. In contrast, however, a number of genes for the archaeal transcription apparatus are organized in clusters resembling the clusters of transcription-associated genes in Bacteria.