Early Stage Of Transcription Research Articles

Alternate Roles of Sox Transcription Factors beyond Transcription Initiation.

Sox proteins are known as crucial transcription factors for many developmental processes and for a wide range of common diseases. They were believed to specifically bind and bend DNA with other transcription factors and elicit transcriptional activation or repression activities in the early stage of transcription. However, their functions are not limited to transcription initiation. It has been showed that Sox proteins are involved in the regulation of alternative splicing regulatory networks and translational control. In this review, we discuss the current knowledge on how Sox transcription factors such as Sox2, Sry, Sox6, and Sox9 allow the coordination of co-transcriptional splicing and also the mechanism of SOX4-mediated translational control in the context of RNA polymerase III.

Magnesium Mediated Fluoride Binding Anchors the Docked Conformation of the Fluoride Riboswitch

Riboswitches are structured non-coding RNA elements typically found in the 5' untranslated regions of bacterial mRNAs. They regulate gene expression, most often at the level of transcription and translation, through the binding of specific ligands that induce changes in the riboswitch structure. Fluoride riboswitches are a family of transcriptional riboswitches that are present in many bacteria and archaea; regulate transcription by preventing the formation of an intrinsic terminator in presence of fluoride. Previous studies have shed light on its structure and the importance of the transcriptional machinery for riboswitch folding, a deep conformational analysis at different points during transcription are needed to fully understand the impact of ligand-binding during transcription. Therefore, we explored the conformational dynamics of the Bacillus cereus crcB fluoride riboswitch at the single-molecule level for both isolated RNA and active transcription elongation complexes. We resolved three conformational states: two undocked and one pseudoknot-structured docked state, and determined their fractional population and interconversion rates. The first undocked state was observed at early stage of transcription; while the other two states, a docked and an undocked state, appear when the aptamer is fully transcribed by RNA polymerase. The riboswitch folds into a transient dynamic docked conformation in presence of magnesium, which upon further binding of fluoride folds into a stably docked conformation. This feature is lost upon disrupting a long-range base pair A40-U48. In active transcription elongation complexes, RNA polymerase fine-tunes the activation energy and promotes the riboswitch to fold into the docked conformation. Further, we showed that ligand binding at short transcript lengths is an early step on the pathway to the docked conformation. These results demonstrate a multi-step mechanism of magnesium- and ligand-dependent conformational transitions that enable regulation of downstream gene expression.

The Role of Pyrophosphorolysis in the Initiation-to-Elongation Transition by E. coli RNA Polymerase

RNA polymerase can cleave a phosphodiester bond at the 3′ end of a nascent RNA in the presence of pyrophosphate producing NTP. Pyrophosphorolysis has been characterized during elongation steps of transcription where its rate is significantly slower than the forward rate of NMP addition. In contrast, we report here that pyrophosphorolysis can occur in a millisecond time scale during the transition of Escherichia coli RNA polymerase from initiation to elongation at the psbA2 promoter. This rapid pyrophosphorolysis occurs during productive RNA synthesis as opposed to abortive RNA synthesis. Dissociation of σ70 or RNA extension beyond nine nucleotides dramatically reduces the rate of pyrophosphorolysis. We argue that the rapid pyrophosphorolysis allows iterative cycles of cleavage and re-synthesis of the 3′ phosphodiester bond by the productive complexes in the early stage of transcription. This iterative process may provide an opportunity for the σ70 to dissociate from the RNA exit channel of the enzyme, enabling RNA to extend through the channel.

Functional interaction of human Ssu72 with RNA polymerase II complexes

Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.

Kinetics of self-assembly via facilitated diffusion: Formation of the transcription complex.

We present an analytically solvable model for self-assembly of a molecular complex on a filament. The process is driven by a seed molecule that undergoes facilitated diffusion, which is a search strategy that combines diffusion in three dimensions and one dimension. Our study is motivated by single-molecule-level observations revealing the dynamics of transcription factors that bind to the deoxyribonucleic acid at early stages of transcription. We calculate the probability that a complex made up of a given number of molecules is completely formed, as well as the distribution of completion times, upon the binding of a seed molecule at a target site on the filament (without explicitly modeling the three-dimensional diffusion that precedes binding). We compare two different mechanisms of assembly where molecules bind in sequential and random order. Our results indicate that while the probability of completion is greater for random binding, the completion time scales exponentially with the size of the complex; in contrast, it scales as a power law or slower for sequential binding, asymptotically. Furthermore, we provide model predictions for the dissociation and residence times of the seed molecule, which are observables accessible in single-molecule tracking experiments.

Revisiting the Function of CDK7 in Transcription by Virtue of a Recently Described TFIIH Kinase Inhibitor

In this issue of Molecular Cell, Nilson etal. (2015) took advantage of THZ1, a recently described covalent inhibitor of the TFIIH kinase CDK7, to further characterize the role of this enzyme in the early stages of transcription and postprocessing events. They unveiled an unexpected function of CDK7 in RNA polymerase II pausing and mRNA capping.

Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I.

Chromatin structure and its alteration play critical roles in the regulation of transcription. However, the transcriptional silencing mechanism with regard to the chromatin structure at an unstimulated state of the interferon (IFN)-stimulated gene (ISG) remains unclear. Here we investigated the role of template activating factor-I (TAF-I, also known as SET) in ISG transcription. Knockdown (KD) of TAF-I increased ISG transcript and simultaneously reduced the histone H1 level on the ISG promoters during the early stages of transcription after IFN stimulation from the unstimulated state. The transcription factor levels on the ISG promoters were increased in TAF-I KD cells only during the early stages of transcription. Furthermore, histone H1 KD also increased ISG transcript. TAF-I and histone H1 double KD did not show the additive effect in ISG transcription, suggesting that TAF-I and histone H1 may act on the same regulatory pathway to control ISG transcription. In addition, TAF-I KD and histone H1 KD affected the chromatin structure near the ISG promoters. On the basis of these findings, we propose that TAF-I and its target histone H1 are key regulators of the chromatin structure at the ISG promoter to maintain the silent state of ISG transcription.

RAM function is dependent on Kapβ2-mediated nuclear entry

Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5′ to 5′ triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5′-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.

Nuclear actin and myosins at a glance

Actin and myosin II form the archetypical molecular motor complex. Myosin II, like all members of the myosin superfamily, is an actin-activated ATPase that uses the energy released when ATP is hydrolyzed to do work. Although we typically associate work with muscle contraction, cell motility and cell

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro

BackgroundCyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. MethodsCyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. ResultsCyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. ConclusionsCyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. General significanceThese data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy.

New Insights into the Mechanism of Initial Transcription

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

Cellular dynamics of the negative transcription elongation factor NELF

Negative Elongation Factor (NELF) is a transcription factor discovered based on its biochemical activity to suppress transcription elongation, and has since been implicated in various diseases ranging from neurological disorders to cancer. Besides its role in promoter-proximal pausing of RNA polymerase II during early stages of transcription, recently we found that it also plays important roles in the 3′-end processing of histone mRNA. Furthermore, NELF has been found to form a distinct subnuclear structure, which we named NELF bodies. These recent developments point to a wide range of potential functions for NELF, and, as most studies on NELF thus far had been carried out in vitro, here, we prepared a complete set of fusion protein constructs of NELF subunits and carried out a general cell biological study of the intracellular dynamics of NELF. Our data show that NELF subunits exhibit highly specific subcellular localizations, such as in NELF bodies or in midbodies, and some shuttle actively between the nucleus and cytoplasm. We further show that loss of NELF from cells can lead to enlarged and/or multiple nuclei. This work serves as a foundation and starting point for further cell biological investigations of NELF in the future.

TRAP-5′ stem–loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region

TRAP regulates expression of the Bacillus subtilis trpEDCFBA operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Bound TRAP blocks formation of an antiterminator structure and allows formation of an overlapping intrinsic terminator upstream of the trp operon structural genes. A 5' stem-loop (5'SL) structure located upstream of the triplet repeat region also interacts with TRAP. TRAP-5'SL RNA interaction participates in the transcription attenuation mechanism by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. Footprinting assays indicated that the 5'SL contacts TRAP through two discrete groups of single-stranded nucleotides that lie in the hairpin loop and in an internal loop. Filter binding and in vivo expression assays of 5'SL mutants established that G7, A8, and A9 from the internal loop, and A19 and G20 from the hairpin loop are critical for proper 5'SL function. These nucleotides are conserved among certain other 5'SL-containing organisms. Single-round transcription results indicated that the 5'SL increases the termination efficiency when transcription is fast; however, the influence of the 5'SL was lost when transcription was slowed by reducing the ribonucleoside triphosphate concentration. Since there is a limited amount of time for TRAP to bind to the nascent transcript and promote termination, our data suggest that the contribution of TRAP-5'SL interaction increases the rate of TRAP binding, which, in turn, increases the efficiency of transcription termination.

The Transition to an Elongation Complex by T7 RNA Polymerase Is a Multistep Process

During the transition from an initiation complex to an elongation complex (EC), T7 RNA polymerase undergoes major conformational changes that involve reorientation of a "core" subdomain as a rigid body and extensive refolding of other elements in the 266 residue N-terminal domain. The pathway and timing of these events is poorly understood. To examine this, we introduced proline residues into regions of the N-terminal domain that become alpha-helical during the reorganization and changed the charge of a key residue that interacts with the RNA:DNA hybrid 5 bp upstream of the active site in the EC but not in the initiation complex. These alterations resulted in a diminished ability to make products >5-7 nt and/or a slow transition through this point. The results indicate that the transition to an EC is a multistep process and that the movement of the core subdomain and reorganization of certain elements in the N-terminal domain commence prior to promoter release (at 8-9 nt).

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon alpha-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene.

Strong natural pausing by RNA polymerase II within 10 bases of transcription start may result in repeated slippage and reextension of the nascent RNA.

We find that immediately following transcript initiation, RNA polymerase II pauses at several locations even in the presence of relatively high (200 microM) levels of nucleoside triphosphates. Strong pauses with half-lives of >30 s were observed at +7, +18/19, and about +25 on the template used in these experiments. We show that the strong pause at +7, after the synthesis of 5'-ACUCUCU, leads to repeated cycles of upstream slippage of the RNA-DNA hybrid followed by re-pairing with the DNA and continued RNA synthesis. The resulting transcripts are 2, 4, and 6 bases longer than predicted by the template sequence. Slippage is efficient when transcription is primed with the +1/+2 (ApC) dinucleotide, and it occurs at even higher levels with the +2/+3 primer (CpU). Slippage can occur at high levels with ATP initiation, but priming with CpA (-1/+1) supports very little slippage. This latter result is not simply an effect of transcript length at the point of pausing. Slippage can also occur with a second template on which the polymerase can be paused after synthesizing ACUCU. Slippage is not reduced by an ATP analog that blocks promoter escape, but it is inhibited by substitution of 5Br-U for U in the RNA. Our results reveal an unexpected flexibility of RNA polymerase II ternary complexes during the very early stage of transcription, and they suggest that initiation at different locations within the same promoter gives rise to transcription complexes with different properties.

The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance.

During the early stages of transcription, T7 RNA polymerase forms an unstable initiation complex that synthesizes and releases transcripts 2-8 nt in length before disengaging from the promoter and isomerizing to a stable elongation complex. In this study, we used RNA small middle dotprotein and RNA small middle dotDNA crosslinking methods to probe the location of newly synthesized RNA in halted elongation complexes. The results indicate that the RNA in an elongation complex remains in an RNA small middle dotDNA hybrid for about 8 nt from the site of nucleotide addition and emerges to the surface of the enzyme about 12 nt from the addition site. Strikingly, as the transcript leaves its hybrid with the template, the crosslinks it forms with the RNA polymerase involve a portion of a hairpin loop (the specificity loop) that makes specific contacts with the binding region of the promoter during initiation. This observation suggests that the specificity loop may have a dual role in transcription, binding first to the promoter and subsequently interacting with the RNA product. It seems likely that association of the nascent RNA with the specificity loop facilitates disengagement from the promoter and is an important part of the process that leads to a stable elongation complex.

HnRNP U inhibits carboxy-terminal domain phosphorylation by TFIIH and represses RNA polymerase II elongation.

This study describes a potential new function of hnRNP U as an RNA polymerase (Pol II) elongation inhibitor. We demonstrated that a subfraction of human hnRNP U is associated with the Pol II holoenzyme in vivo and as such recruited to the promoter as part of the preinitiation complex. hnRNP U, however, appears to dissociate from the Pol II complex at the early stage of transcription and is therefore absent from the elongating Pol II complex. When tested in the human immunodeficiency virus type 1 transcription system, hnRNP U inhibits elongation rather than initiation of transcription by Pol II. This inhibition requires the carboxy-terminal domain (CTD) of Pol II. We showed that hnRNP U can bind TFIIH in vivo under certain conditions and inhibit TFIIH-mediated CTD phosphorylation in vitro. We find that the middle domain of hnRNP U is sufficient to mediate its Pol II association and its inhibition of TFIIH-mediated phosphorylation and Pol II elongation. The abilities of hnRNP U to inhibit TFIIH-mediated CTD phosphorylation and its Pol II association are necessary for hnRNP U to mediate the repression of Pol II elongation. Based on these observations, we suggest that a subfraction of hnRNP U, as a component of the Pol II holoenzyme, may downregulate TFIIH-mediated CTD phosphorylation in the basal transcription machinery and repress Pol II elongation. With such functions, hnRNP U might provide one of the mechanisms by which the CTD is maintained in an unphosphorylated state in the Pol II holoenzyme.

Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases.

We have used UV-laser mediated cross-linking, DNase I footprinting and KMnO4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription. In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, and +3 and on the nontemplate (NT) strand at position -8. These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T strand and from positions -17 to +16 on the NT strand and straddle sites of enhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand. Use of supercoiled plasmid templates has allowed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time. Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products. Under these conditions the downstream contact on the T strand switched from position +3 to +4 and +5 while the contact at position -17 was maintained. Under conditions in which the synthesis of transcription products is limited to 6-7 nucleotides, only the contact at position -17 on the T strand was preserved. A comparison of these results with the interaction of Escherichia coli RNA polymerase at the lac promoter reveals strong similarities in the manner in which these polymerases recognize their promoters.

A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription

We have identified a mutation (E148A) in T7 RNA polymerase (RNAP) that results in an enzyme which aborts transcription primarily when the nascent RNA achieves a length of 5 nt. This phenomenon is observed at a consensus promoter, but is even more strongly observed at promoters that are altered in the initiation region. Although the abortive product is of a fixed length (5 nt), the positions of the base substitutions in the initiation region that enhance this effect do not appear to be fixed, and we have observed the effect with a variety of initiation-region promoter variants. The phenomenon is also observed during promoter-independent transcription when transcribing a homopolymeric template such as poly(dC). Under conditions where the active site of the RNAP cannot extend beyond the third nucleotide in the template strand and the maximum length of the RNA:DNA hybrid cannot exceed three base-pairs (i.e. when synthesizing oligoG products due to transcript slippage at a promoter that initiates with the sequence +1 GGG.) the mutant RNAP gives rise to a normal spectrum of products 2 to 14 nt in length with no evidence of a block at 5 nt. Neither promoter binding nor promoter melting appears to be involved in this phenotype, as the mutant RNAP binds normally to promoter sequences and the behavior of the enzyme is unaffected by removal of the non-template strand in the initiation region of the promoter or on a supercoiled template. Importantly, the mutant RNAP is defective in binding single strand oligomers of RNA. These results suggest that the affected region of the RNAP may form part of the RNA product binding site and may be involved in the transition from an unstable initiation complex to a stable elongation complex, perhaps by sensing the presence of a nascent RNA and/or RNA:DNA hybrid.