Abortive RNA Synthesis Research Articles

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.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation.

Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has ‘post-recruitment’ roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from −4 to +1 in the open complex with all three proteins and from −4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar Kd values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery.

Replication-Coupled and Host Factor-Mediated Encapsidation of the Influenza Virus Genome by Viral Nucleoprotein

The influenza virus RNA-dependent RNA polymerase is capable of initiating replication but mainly catalyzes abortive RNA synthesis in the absence of viral and host regulatory factors. Previously, we reported that IREF-1/minichromosome maintenance (MCM) complex stimulates a de novo initiated replication reaction by stabilizing an initiated replication complex through scaffolding between the viral polymerase and nascent cRNA to which MCM binds. In addition, several lines of genetic and biochemical evidence suggest that viral nucleoprotein (NP) is involved in successful replication. Here, using cell-free systems, we have shown the precise stimulatory mechanism of virus genome replication by NP. Stepwise cell-free replication reactions revealed that exogenously added NP free of RNA activates the viral polymerase during promoter escape while it is incapable of encapsidating the nascent cRNA. However, we found that a previously identified cellular protein, RAF-2p48/NPI-5/UAP56, facilitates replication reaction-coupled encapsidation as an NP molecular chaperone. These findings demonstrate that replication of the virus genome is followed by its encapsidation by NP in collaboration with its chaperone.

Real-Time Initial Transcription by a Multisubunit RNA Polymerase

Abortive initiation, the repetitive synthesis of short RNA transcripts before promoter escape and elongation, is a highly dynamic process that can constitute the rate-limiting step for many gene promoters. Previous studies using single-molecule Förster Resonance Energy Transfer (smFRET) demonstrated that abortive initiation occurs via a DNA-scrunching mechanism mediated by RNA polymerase. While these studies, along with biochemical work, have yielded insight into abortive initiation, no single-molecule method has been able to directly capture RNA synthesis/release, or the associated conformational changes of the transcription complex. Here, we descibe a highly sensitive smFRET assay that, for the first time, monitors transcription bubble expansion and compaction during abortive RNA synthesis and release in real-time; we also performed similar analysis using an assay that reports on the movement of downstream DNA toward the RNA polymerase main channel. Using single-molecule time trajectories, we have observed multiple cycles (up to 50) of RNA synthesis by single RNA polymerase molecules and we used them to obtain the rates of RNA synthesis and release, as well as to study the dependence of the rates on nucleotide concentration and promoter sequence. Our initial results revealed that, surprisingly, the rate of nucleotide addition in initial transcription is 5-10 fold faster than elongation; moreover, we observed large heterogeneity between different RNA polymerase molecules with regards to specific steps in initial transcription; such heterogeneity may contribute to the noise associated with gene expression.

Transcription Initiation in a Single-Subunit RNA Polymerase Proceeds through DNA Scrunching and Rotation of the N-Terminal Subdomains

Elucidating the mechanism of transcription initiation by RNA polymerases (RNAP) is essential for understanding gene transcription and regulation. Although several models, such as DNA scrunching, RNAP translation, and RNAP rotation, have been proposed, the mechanism of initiation by T7 RNAP has remained unclear. Using ensemble and single-molecule Förster resonance energy transfer (FRET) studies, we provide evidence for concerted DNA scrunching and rotation during initiation by T7 RNAP. A constant spatial distance between the upstream and downstream edges of initiation complexes making 4-7 nt RNA supports the DNA scrunching model, but not the RNAP translation or the pure rotation model. DNA scrunching is accompanied by moderate hinging motion (18 degrees +/- 4 degrees ) of the promoter toward the downstream DNA. The observed stepwise conformational changes provide a basis to understand abortive RNA synthesis during early stages of initiation and promoter escape during the later stages that allows transition to processive elongation.

LacP1 Promoter with an Extended – 10 Motif: PLEIOTROPIC EFFECTS OF CYCLIC AMP PROTEIN AT DIFFERENT STEPS OF TRANSCRIPTION INITIATION

The cyclic AMP receptor protein (CRP), which activates transcription from the wild-type lacP1 promoter and most of its mutants, represses productive RNA synthesis from a lacP1 promoter variant that contains an extended -10 element, although CRP enhances RNA polymerase binding as well as open complex formation in both promoters. Moreover, abortive RNA synthesis, which is already higher in the extended -10 variant compared with the parent promoter, was further enhanced by CRP. These results, together with the observed decrease in productive RNA synthesis, indicate that CRP, while facilitating the earlier steps of initiation, inhibits transcription from the extended -10 lacP1 by hindering promoter clearance. We propose that CRP decreases energetic barriers to RNA polymerase binding, isomerization, and abortive RNA synthesis but stabilizes the abortive RNA initiating complex, which results in increasing the activation energy of the transition state before the elongation complex. The results demonstrate for the first time that a DNA-binding regulatory protein acts as an activator or a repressor in different steps of the transcription initiation pathway because of the energetic differences of the intermediate complex in the same promoter.

Effect of varying the supercoiling of DNA on transcription and its regulation.

The effect of superhelicity of DNA templates on transcription is well documented in several cases. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such effects are exerted were not systematically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters present on a plasmid by using a series of topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays with these topoisomers in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter to promoter. Some of those promoters, in which DNA superhelicity stimulated transcription, displayed specific optima of superhelical density while others did not. The results also showed that the amounts of abortive RNA synthesis from two of the promoters decreased and full-length RNA increased with increasing supercoiling, indicating for the first time an inverse relationship between full-length and abortive RNA synthesis and supporting a role of DNA superhelicity in promoter clearance. DNA supercoiling might also influence the point of RNA chain termination. Furthermore, the effect of varying the amount of supercoiling on the action of gene regulatory proteins suggested the mode of action, which is consistent with previous results. Our results underscore the importance of DNA supercoiling in fine-tuning promoter activities, which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments.

Reduction in Abortive Transcription from the λPR Promoter by Mutations in Region 3 of the ς70 Subunit of Escherichia coli RNA Polymerase

Transcription initiation by Escherichia coli RNA polymerase at most promoters is associated with a reiterative synthesis and release of short abortive RNA products. We have investigated the mechanism of abortive RNA synthesis by using holoenzymes containing mutant sigma70 subunits with changes in region 3 (S506F and P504L), which reduce the ratio of abortive to full-length products. Binary complexes formed by these mutant enzymes at a modified lambdaPR promoter contained a smaller fraction of open complexes than for normal polymerase, suggesting an involvement of region 3 in melting duplex DNA or in maintenance of the open complex. The half-lives of the majority of binary complexes formed by the mutant enzymes were less than 1 min, in contrast to 30 min for the wild-type complexes. The time courses of transcription and pulse-labeling assays showed that moribund complexes, which generate only abortive products (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457), were formed by the mutant enzymes. However, they accumulated to a lesser extent than for the wild-type enzyme, due both to faster dissociation and conversion into inactive complexes. This is the main cause of the low degree of abortive transcription displayed by the mutant enzymes on this promoter.

Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase.

The kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase was investigated using transient state kinetic methods. Transcription by bacteriophage T7 RNA polymerase occurs in three stages consisting of initiation, promoter clearance, and elongation. Abortive products, up to 6-8-mer, were synthesized during the initiation phase; the transition from initiation to elongation occurred between the synthesis of 6-8-mer and 11-12-mer, and the processive elongation phase began after the synthesis of 12-mer RNA. Our results show that the synthesis of elongation product from the phi 10 promoter is limited both by the efficiency of initiation and by the frequency at which the polymerase escapes the promoter. Studies with heparin trap suggest that the polymerase maintains contact with the promoter region during multiple turnovers of abortive RNA synthesis; thus, the polymerase does not completely dissociate from the promoter after each event of abortive RNA synthesis. The pre-steady-state kinetics of RNA synthesis indicate that initiation occurs at a rate constant (3.5 s(-1)) that is about 30 times faster than the steady-state rate constant of RNA synthesis (0.1 s(-1)). The steady-state rate constant of RNA synthesis is limited largely by the cycling of the RNA polymerase, whereas initiation is limited by the formation of pppGpG, the first RNA product. We show that the synthesis of pppGpG is not limited by steps associated with GTP binding, DNA binding, or the melting of the promoter DNA. Instead, the kinetic results indicate that initiation at the phi10 promoter is limited either by the first phosphodiester bond formation step or more likely by a conformational change prior to pppGpG formation. Such a conformational change could play a role in proper alignment of the initiating and elongating NTPs for efficient phosphodiester bond formation and in maintaining the fidelity of RNA synthesis.

Effect of guanosine tetraphosphate (ppGpp) on the conformational state ofE. coli RNA polymerase and transcription directed by tyrT, T7D, and T7A1 promoters

Guanosine tetraphosphate (ppGpp) is an allosteric regulator of DNA-dependent RNA polymerase. It selectively inhibits transcription from some promoters containing the GC-rich sequence near the starting point of transcription. Using the method of the fluorescent label it was shown that interacting with the β-subunit of the enzyme ppGpp induces essential conformational transitions of the RNA polymerase. The mutation in the enzyme β-subunit that increases the polymerase affinity for T7D promoter affects ppGpp-induced conformational changes. The rate of the abortive RNA synthesis directed by the mutant enzyme from this promoter, which contains no GC-rich sequences near the starting point appeared to be ppGpp-dependent. ppGpp has no effect on T7D activity in the system containing the native enzyme. Thus, the regulatory function of ppGpp depends on the structure of the RNA polymerase and could be realized by the enzyme not only by recognition of the GC-rich block in the promoter.

Nucleotide Sequences of Reovirus Oligonucleotides Evidence for Abortive RNA Synthesis during Virus Maturation

IN addition to the ten double stranded segments of genome RNA, reovirions contain many hundreds of single-stranded oligoribonucleotides from 3 to 20 residues long, which fall into two classes. First, those precipitated by acid or ethanol and very rich in adenine1–2; second, those poorly or not precipitable and therefore shorter, such as the trinucleotidepppGpCpUOH3,4. The former class of molecules comprises the fraction previously designated “A-rich RNA”1, which has been shown to be a component of all three serotypes of human reoviruses2, as well as of avian reovirus5, irrespective of the nature of the host cell in which the virus was propagated1. These molecules are synthesized while viral progeny accumulate within the cell1; they do not appear free in the cytoplasm, but are found only in material which can be pelleted by centrifuging at 20,000g for 30 min1 with which maturing virus particles are also known to be associated6 and the same is probably also true for the second class of oligonucleotides. Although the amount of A-rich oligonucleotides in reovirus is usually constant, the amount of the other oligonucleotides is about five times less in virus grown at 31° C than at 40° C7.