T7 RNA polymerase functions in vitro without clustering.
Ontology highlight
ABSTRACT:
SUBMITTER: Finan K
PROVIDER: S-EPMC3388079 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Publications
T7 RNA polymerase functions in vitro without clustering.
Finan Kieran K Torella Joseph P JP Kapanidis Achillefs N AN Cook Peter R PR
PloS one 20120702 7
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 occ ...[more]
Similar Datasets
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.
| S-EPMC21300 | biostudies-literature
Project description:A class of oligonucleotides which binds to naturally-occurring duplex DNA sites at physiologic pH to form triple helical structures was used as transcription attenuators in an in vitro transcription assay. Oligonucleotides were designed to form triple helices with a purine-rich, double-stranded target by binding in the major groove in an orientation anti-parallel to the most purine-rich strand of the target. A 45 base-pair purine-rich region located within the gag gene of Friend Murine Leukemia Virus (FMLV) was used as the duplex target. The target DNA was inserted by molecular cloning downstream of either the bacterial T7- or T3 promoter. The sequence-specific interaction of the triple helix-forming oligonucleotide (TFO) with the FMLV target was confirmed by DNAse I footprint analysis. The affinity of the TFO, as measured by the equilibrium dissociation constant of the TFO for the duplex, was determined by band shift analysis. When a TFO was allowed to form a triple helix with the target duplex in well-defined buffer conditions before the transcription reaction, truncated transcripts of a predicted size were observed. Attenuation of transcription was observed only when buffer conditions favorable to triple helix formation were used. In addition, oligonucleotides containing a high percentage of guanosine residues were able to inhibit mRNA production of the bacterial T7 polymerase by a mechanism independent of transcription attenuation. The ability of an oligonucleotide-directed triple helical structure to slow down, or even completely stop, RNA chain elongation may expand the utility of triple helix technology in the area of gene regulation.
| S-EPMC307860 | biostudies-other
Project description:Escherichia coli rrnB terminator t1 contains an RNA hairpin-dependent (class I) and a sequence-specific (class II) termination signal. The latter consists of an 8-bp conserved sequence (CS), TATCTGTT, immediately followed by an 8-bp T rich sequence. In this study, elongation complexes of T7 RNA polymerase at various positions of the class II signal and several mutant signals were obtained by stepwise walking on immobilized DNA templates free of the class I signal. Multiple CS-associated conformational changes were observed, starting at the beginning of the signal and occurring sequentially. When the complexes reach the first base pair of the CS-DNA duplex, which is downstream of the RNA-DNA heteroduplex, their stability, as measured by time-course retention of radiolabeled transcripts, markedly decreases. Further elongation leads to an abrupt change in polymerase-RNA interaction. Cross-linking of the polymerase to a 4-thio-UMP incorporated into RNA 8 nucleotides upstream of the 3' end and just upstream of the heteroduplex is initially strong but diminishes when the polymerase reaches the fourth base pair of the CS. After a further 7-nt elongation, the exposed single-stranded region of nontemplate strand is contracted; RNA in the upstream half of the heteroduplex becomes dissociated, and the CS-DNA duplex is reformed. During the next 5-nt elongation before termination, the CS duplex is prevented from translocation, and the contracted transcription bubble expands only downstream. These findings suggest that the CS duplex plays essential roles by successively binding to polymerase both downstream and upstream of the heteroduplex.
| S-EPMC544054 | biostudies-literature
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.
| S-EPMC7668259 | biostudies-literature
Project description:Engineering protein expression in vitro or in vivo is usually straightforward for single genes, but remains challenging for multiple genes because of the requirement of coordinated control. RNA and protein overexpression strategies often exploit T7 RNA polymerase and its natural TPhi Class I terminator. However, this terminator's inefficiency and large size (100 bp) are problematic for multigene construction and expression. Here, we measure the effects of tandem copies of a small (18 bp) Class II T7 terminator from vesicular stomatitis virus on transcription in vitro and on translation in vitro and in vivo. We first test monomeric and dimeric gene constructs, then attempt extension to pentameric gene constructs. "BioBrick" versions of a pET vector and translation factor genes were constructed to facilitate cloning, and His-tags were incorporated to allow copurification of all protein products for relatively unbiased analysis and easy purification. Several results were surprising, including imbalanced expression of the pentameric constructs in vivo, illustrating the value of synthetic biology for investigating gene expression. However, these problems were solved rationally by changing the orders of the genes and by adding extra promoters to the upstream gene or by moving to a more predictable in vitro translation system. These successes were significant, given our initial unexpected results and that we are unaware of another example of coordinated overexpression of five proteins. Our modular, flexible, rational method should further empower synthetic biologists wishing to overexpress multiple proteins simultaneously.
| S-EPMC2783478 | biostudies-literature
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.
| S-EPMC3642155 | biostudies-literature
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.
| S-EPMC1986751 | biostudies-literature
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.
| S-EPMC1170743 | biostudies-other
Project description:Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the "Broccoli" RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.
| S-EPMC7948468 | biostudies-literature
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