Wild-type RNA Polymerase Research Articles

DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants.

The remarkable success of messenger RNA (mRNA)-based vaccines has underscored their potential as a novel biotechnology platform for vaccine development and therapeutic protein delivery. However, the single-subunit RNA polymerase from bacteriophage T7 widely used for in vitro transcription is well known to generate double-stranded RNA (dsRNA) by-products that strongly stimulate the mammalian innate immune response. The dsRNA was reported to be originated from self-templated RNA extension or promoter-independent transcription. Here, we identified that the primary source of the full-length dsRNA during in vitro transcription is the DNA-terminus-initiated transcription by T7 RNA polymerase. Guanosines or cytosines at the end of DNA templates enhance the DNA-terminus-initiated transcription. Moreover, we found that aromatic residues located at position 47 in the C-helixleadto a significant reduction in the production of full-length dsRNA. As a result, the mRNA synthesized using the T7 RNA polymerase G47W mutant exhibits higher expression efficiency and lower immunogenicity compared to the mRNA produced using the wild-type T7 RNA polymerase.

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst.

Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature. To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used. The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.

An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis.

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

Heat Treatment Promotes Ubiquitin-Mediated Proteolysis of SARS-CoV-2 RNA Polymerase and Decreases Viral Load

Despite extensive efforts, COVID-19 pandemic caused by the SARS-CoV-2 virus is still at large. Vaccination is an effective approach to curb virus spread, but several variants (e.g., delta, delta plus, omicron, and IHU) appear to weaken or possibly escape immune protection. Thus, novel and quickly scalable approaches to restrain SARS-CoV-2 are urgently needed. Multiple evidences showed thermal sensitivity of SARS-CoV-2 and negative correlation between environmental temperature and COVID-19 transmission with unknown mechanism. Here, we reveal a potential mechanism by which mild heat treatment destabilizes the wild-type RNA-dependent RNA polymerase (also known as nonstructural protein 12 (NSP12)) of SARS-CoV-2 as well as the P323L mutant commonly found in SARS-CoV-2 variants, including omicron and IHU. Mechanistically, heat treatment promotes E3 ubiquitin ligase ZNF598-dependent NSP12 ubiquitination leading to proteasomal degradation and significantly decreases SARS-CoV-2 RNA copy number and viral titer. A mild daily heat treatment maintains low levels of both wild-type and P323L mutant of NSP12, suggesting clinical potential. Collectively, this novel mechanism, heat-induced NSP12 degradation, suggests a prospective heat-based intervention against SARS-CoV-2.

Uncovering the Resistance Mechanism of Mycobacterium tuberculosis to Rifampicin Due to RNA Polymerase H451D/Y/R Mutations From Computational Perspective.

Tuberculosis is still one of the top 10 causes of deaths worldwide, especially with the emergence of multidrug-resistant tuberculosis. Rifampicin, as the most effective first-line antituberculosis drug, also develops resistance due to the mutation on Mycobacterium tuberculosis (Mtb) RNA polymerase. Among these mutations, three mutations at position 451 (H451D, H451Y, H451R) are associated with high-level resistance to rifampicin. However, the resistance mechanism of Mtb to rifampicin is still unclear. In this work, to explore the resistance mechanism of Mtb to rifampicin due to H451D/Y/R mutations, we combined the molecular dynamics simulation, molecular mechanics generalized-Born surface area calculation, dynamic network analysis, and residue interactions network analysis to compare the interaction change of rifampicin with wild-type RNA polymerase and three mutants. The results of molecular mechanics generalized-Born surface area calculations indicate that the binding free energy of rifampicin with three mutants decreases. In addition, the dynamic network analysis and residue interaction network analysis show that when H451 was mutated, the interactions of residue 451 with its adjacent residues such as Q438, F439, M440, D441, and S447 disappeared or weakened, increasing the flexibility of binding pocket. At the same time, the disappearance of hydrogen bonds between R613 and rifampicin caused by the flipping of R613 is another important reason for the reduction of binding ability of rifampicin in H451D/Y mutants. In H451R mutant, the mutation causes the binding pocket change too much so that the position of rifampicin has a large movement in the binding pocket. In this study, the resistance mechanism of rifampicin at the atomic level is proposed. The proposed drug-resistance mechanism will provide the valuable guidance for the design of antituberculosis drugs.

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

RNA cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA cleavage. RNA cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli, Deinococcus radiodurans, and Thermus aquaticus, we show that the functions of the TL and Gre factors in RNA cleavage are conserved in various species, with important variations that may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA cleavage by all three RNAPs and eliminate the interspecies differences in the reaction rates. GreA factors activate RNA cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent cleavage are lower for ΔTL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA cleavage in certain types of backtracked transcription complexes, suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.

Negative selection and stringency modulation in phage-assisted continuous evolution.

Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to dramatically accelerate laboratory evolution experiments. In this work we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counter-selection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates, while maintaining wild-type-like levels of activity.

Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags

Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3' homogeneity. Here, we explored strategies to also ensure 5' homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II 2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5'-sequence heterogeneity in several cases, significant levels of 5' heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5' homogeneity, we tested the suitability of using a small CRISPR RNA stem-loop at the 5' end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5'-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR-SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5'-CRISPR-based strategy can be combined with affinity purification using the 3'-ARiBo tag for quick purification of RNA with both 5' and 3' homogeneity.

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryptophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3'-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.

Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation.

Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of λP(R) promoter DNA to form the unstable open intermediate I(2), which rapidly converts to much more stable open complexes (I(3), RP(o)). Conversion of I(2) to RP(o) involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile β' jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable ΔJAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5-10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with ΔJAW RNAP and/or DT+12 DNA resemble those deduced for the structurally uncharacterized intermediate I(3). Overall rate constants (k(a)) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of k(a) for WT RNAP with DT+12 and full-length λP(R) are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RP(o) formation, while downstream DNA functions primarily as the assembly platform after DNA opening.

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.

Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

Citrus paradisi Macfad. ‘Duncan’ was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from a CTV genome and used to generate the wild-type and 2 mutant RdRp constructs for plant transformation. One mutant had the key amino acids GDD changed to AAA (RdRp-mGDD), and the second mutant had a deletion encompassing the GDD domain (RdRp-DGDD). Etiolated epicotyl segments of Duncan grapefruit (Citrus paradisi Macfad. 'Duncan') were transformed with each of these constructs using the Agrobacterium-mediated transformation method. From 4540 transformed epicotyl segments, 1402 kanamycin-resistant shoots were regenerated. After testing for expression of green fluorescent protein (GFP) and \Beta-glucuronidase (GUS) reporter genes by fluorescence microscopy and histochemical staining, respectively, 146 GUS-positive plants were rooted and 97 surviving plants were established in soil in pots. A total of 70 plants were tested for the presence of the GUS gene and CTV RdRp transgenes by polymerase chain reaction (PCR). A total of 51 GUS and CTV transgene-positive transgenic plants (15 with RdRp, 21 with RdRp-mGDD, and 15 with RdRp-DGDD) were identified.

Interaction of Escherichia coli RNA Polymerase σ70 Subunit with Promoter Elements in the Context of Free σ70, RNA Polymerase Holoenzyme, and the β′-σ70 Complex

Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ(70) from Escherichia coli, the β'-σ complex, and the σ(70) RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ(70), free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ(70) is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β' RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ(70). The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

Maintenance of RNA-DNA Hybrid Length in Bacterial RNA Polymerases

During transcription elongation the nascent RNA remains base-paired to the template strand of the DNA before it is displaced and the two strands of the DNA reanneal, resulting in the formation of a transcription "bubble" of approximately 10 bp. To examine how the length of the RNA-DNA hybrid is maintained, we assembled transcription elongation complexes on synthetic nucleic acid scaffolds that mimic the situation in which transcript displacement is compromised and the polymerase synthesizes an extended hybrid. We found that in such complexes bacterial RNA polymerase exhibit an intrinsic endonucleolytic cleavage activity that restores the hybrid to its normal length. Mutations in the region of the RNA polymerase near the site of RNA-DNA separation result in altered RNA displacement and translocation functions and as a consequence in different patterns of proofreading activities. Our data corroborate structural findings concerning the elements involved in the maintenance of the length of the RNA-DNA hybrid and suggest interplay between polymerase translocation, DNA strand separation, and intrinsic endonucleolytic activity.

Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature‐sensitive

All archaeal genomes encode RNA polymerase (RNAP) subunits E and F that share a common ancestry with the eukaryotic RNAP subunits A43 and A14 (Pol I), Rpb7 and Rpb4 (Pol II), and C25 and C17 (Pol III). By gene replacement, we have isolated archaeal mutants of Thermococcus kodakarensis with the subunit F-encoding gene (rpoF) deleted, but we were unable to isolate mutants lacking the subunit E-encoding gene (rpoE). Wild-type T. kodakarensis grows at temperatures ranging from 60 degrees C to 100 degrees C, optimally at 85 degrees C, and the DeltarpoF cells grew at the same rate as wild type at 70 degrees C, but much slower and to lower cell densities at 85 degrees C. The abundance of a chaperonin subunit, CpkB, was much reduced in the DeltarpoF strain growing at 85 degrees C and increased expression of cpkB, rpoF or rpoE integrated at a remote site in the genome, using a nutritionally regulated promoter, improved the growth of DeltarpoF cells. RNAP preparations purified from DeltarpoF cells lacked subunit F and also subunit E and a transcription factor TFE that co-purifies with RNAP from wild-type cells, but in vitro, this mutant RNAP exhibited no discernible differences from wild-type RNAP in promoter-dependent transcription, abortive transcript synthesis, transcript elongation or termination.

Transcriptional pausing and stalling causes multiple clustered mutations by human activation‐induced deaminase

Transcription of the rearranged immunoglobulin gene and expression of the enzyme activation-induced deaminase (AID) are essential for somatic hypermutations of this gene during antibody maturation. While AID acts as a single-strand DNA-cytosine deaminase creating U . G mispairs that lead to mutations, the role played by transcription in this process is less clear. We have used in vitro transcription of the kan gene by the T7 RNA polymerase (RNAP) in the presence of AID and a genetic reversion assay for kanamycin-resistance to investigate the causes of multiple clustered mutations (MCMs) during somatic hypermutations. We find that, depending on transcription conditions, AID can cause single-base substitutions or MCMs. When wild-type RNAP is used for transcription at physiologically relevant concentrations of ribonucleoside triphosphates (NTPs), few MCMs are found. In contrast, slowing the rate of elongation by reducing the NTP concentration or using a mutant RNAP increases several-fold the percent of revertants containing MCMs. Arresting the elongation complexes by a quick removal of NTPs leads to formation of RNA-DNA hybrids (R-loops). Treatment of these structures with AID results in a high percentage of Kan(R) revertants with MCMs. Furthermore, selecting for transcription elongation complexes stalled near the codon that suffers mutations during acquisition of kanamycin-resistance results in an overwhelming majority of revertants with MCMs. These results show that if RNAP II pauses or stalls during transcription of immunoglobulin gene, AID is likely to promote MCMs. As changes in physiological conditions such as occurrence of certain DNA primary or secondary structures or DNA adducts are known to cause transcriptional pausing and stalling in mammalian cells, this process may cause MCMs during somatic hypermutation.

Allosteric control of the RNA polymerase by the elongation factor RfaH

Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that ‘fast’ RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis–Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 Å away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and λQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex.

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).

Template Misalignment in Multisubunit RNA Polymerases and Transcription Fidelity

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

The Role of the Lid Element in Transcription by E. coli RNA Polymerase

The recently described crystal structures of multi-subunit RNA polymerases (RNAPs) reveal a conserved loop-like feature called the lid. The lid projects from the clamp domain and contacts the flap, thereby enclosing the RNA transcript in RNAP's RNA-exit channel and forming the junction between the exit channel and the main channel, which holds the RNA:DNA hybrid. In the initiating form of bacterial RNAP (holoenzyme containing σ), the lid interacts with σ region 3 and encloses an extended linker between σ region 3 and σ region 4 in place of the RNA in the exit channel. During initiation, the lid may be important for holding open the transcription bubble and may help displace the RNA from the template DNA strand. To test these ideas, we constructed and characterized a mutant RNAP from which the lid element was deleted. Δlid RNAP exhibited dramatically reduced activity during initiation from −35-dependent and −35-independent promoters, verifying that the lid is important for stabilizing the open promoter complex during initiation. However, transcript elongation, RNA displacement, and, surprisingly, transcriptional termination all occurred normally in Δlid RNAP. Importantly, Δlid RNAP behaved differently from wild-type RNAP when transcribing single-stranded DNA templates where it synthesized long, persistent RNA:DNA hybrids, in contrast to efficient transcriptional arrest by wild-type RNAP.