Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP
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ABSTRACT:
SUBMITTER: Pawlowska R
PROVIDER: S-EPMC10905585 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Project description:Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3'-5' proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.
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Project description:Recent models of RNA polymerase transcription complexes have invoked the idea that enzyme-nascent RNA contacts contribute to the stability of the complexes. Although much progress on this topic has been made with the multisubunit Escherichia coli RNA polymerase, there is a paucity of information regarding the structure of single-subunit phage RNA polymerase transcription complexes. Here, we photo-cross-linked the RNA in a T7 RNA polymerase transcription complex and mapped a major contact site between amino acid residues 144 and 168 and probably a minor contact between residues 1 and 93. These regions of the polymerase are proposed to interact with the emerging RNA during transcription because the 5' end of the RNA was cross-linked. The contacts are both ionic and nonionic (hydrophobic). The specific inhibitor of T7 transcription, T7 lysozyme, does not compete with T7 RNA polymerase for RNA cross-linking, implying that the RNA does not bind the lysozyme. However, lysozyme may act indirectly via a conformational change in the polymerase. In the current model, the DNA template lies in the polymerase cleft and the fingers subdomain may contact or maintain a template bubble, and a region in the N terminus forms a partly solvent-accessible binding channel for the emerging RNA.
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Project description:RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as cotranscriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We hypothesized that stalling the polymerase downstream from the promoter region and subsequent cleavage of the promoter by a restriction enzyme (to prevent promoter binding by another polymerase) would allow synchronized production of a single transcript per template. The single-pass transcription was verified in two different scenarios: a short self-cleaving ribozyme and a long mRNA. The results show that a controlled single-pass transcription using T7 RNAP allows precise measurement of cotranscriptional ribozyme activity, and this approach will facilitate the study of other kinetic events.
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Project description:Aptamers are promising gene components that can be used for the construction of synthetic gene circuits. In this study, we isolated an RNA aptamer that specifically inhibits transcription of T7 RNA polymerase (RNAP). The 38-nucleotide aptamer, which was a shortened variant of an initial SELEX isolate, showed moderate inhibitory activity. By stepwise doped-SELEX, we isolated evolved variants with strong inhibitory activity. A 29-nucleotide variant of a doped-SELEX isolate showed 50% inhibitory concentration at 11 nM under typical in vitro transcription conditions. Pull-down experiments revealed that the aptamer inhibited the association of T7 RNAP with T7 promoter DNA.
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Project description:Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using 'pulldowns' and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a K(d)<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein.
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Project description:Bacteriophage T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here, we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysine residues (K711/K713/K714) are present during both elongation and initiation, where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability.
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Project description:The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold.
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Project description:O(6)-Methylguanine (O(6)-meG) is a major mutagenic, carcinogenic and cytotoxic DNA adduct produced by various endogenous and exogenous methylating agents. We report the results of transcription past a site-specifically modified O(6)-meG DNA template by bacteriophage T7 RNA polymerase and human RNA polymerase II. These data show that O(6)-meG partially blocks T7 RNA polymerase and human RNA polymerase II elongation. In both cases, the sequences of the truncated transcripts indicate that both polymerases stop precisely at the damaged site without nucleotide incorporation opposite the lesion, while extensive misincorporation of uracil is observed in the full-length RNA. For both polymerases, computer models suggest that bypass occurs only when O(6)-meG adopts an anti conformation around its glycosidic bond, with the methyl group in the proximal orientation; in contrast, blockage requires the methyl group to adopt a distal conformation. Furthermore, the selection of cytosine and uracil partners opposite O(6)-meG is rationalized with modeled hydrogen-bonding patterns that agree with experimentally observed O(6)-meG:C and O(6)-meG:U pairing schemes. Thus, in vitro, O(6)-meG contributes substantially to transcriptional mutagenesis. In addition, the partial blockage of RNA polymerase II suggests that transcription-coupled DNA repair could play an auxiliary role in the clearance of this lesion.
| S-EPMC2582612 | biostudies-literature
Project description:The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining 2 short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of 3 of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.
| S-EPMC9157156 | biostudies-literature
Project description:BackgroundT7 lysozyme (T7L), also known as N-acetylmuramoyl-L-alanine amidase, is a T7 bacteriophage gene product. It involves two functions: It can cut amide bonds in the bacterial cell wall and interacts with T7 RNA polymerase (T7RNAP) as a part of transcription inhibition. In this study, with the help of molecular dynamics (MD) calculations and computational interaction studies, we investigated the effect of varying pH conditions on conformational flexibilities of T7L and their influence on T7RNAP -T7L interactions.ResultsFrom the MD studies of the T7L at three different pH strengths viz. 5, neutral and 7.9 it was observed that T7L structure at pH 5 exhibited less stable nature with more residue level fluctuations, decrease of secondary structural elements and less compactness as compared to its counterparts: neutral pH and pH 7.9. The T-pad analysis of the MD trajectories identified local fluctuations in few residues that influenced the conformational differences in three pH strengths. From the docking of the minimum energy representative structures of T7L at different pH strengths (obtained from the free energy landscape analysis) with T7RNAP structures at same pH strengths, we saw strong interaction patterns at pH 7.9 and pH 5. The MD analysis of these complexes also confirmed the observations of docking study. From the combined in silico studies, it was observed that there are conformational changes in N-terminal and near helix 1 of T7L at different pH strengths, which are involved in the T7RNAP interaction, thereby varying the interaction pattern.ConclusionSince T7L has been used for developing novel therapeutics and T7RNAP one of the most biologically useful protein in both in-vitro and in vivo experiments, this in silico study of pH dependent conformational differences in T7L and the differential interaction with T7RNAP at different pH can provide a significant insight into the structural investigations on T7L and T7RNAP in varying pH environments.
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