Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA.

Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5'-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5'-triphosphate (ATP) at the subunit interface stabilizes the subunit-subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5'-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein-protein and protein-DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein-ssDNA force field and elucidate the molecular basis of replicative helicase translocation.

Dinucleoside Polyphosphates as RNA Building Blocks with Pairing Ability in Transcription Initiation.

Dinucleoside polyphosphates (NpnNs) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5' RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed NpnNs to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of ApnGs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the -1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.

Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection.

The expression level of miRNA-21 is closely related to the occurrence and development of cancer, especially in gastrointestinal cancer. Monitoring miRNA-21 has clinical application in the diagnosis and evaluation of gastrointestinal cancer. A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for miRNA-21 determination by using carbon dots (CDs) and FAM-labeled ssDNA as the signal source. CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy. In the absence of miRNA-21, FAM-labeled ssDNA would be adsorbed and quenched by CDs. The addition of miRNA-21 induced cycle hydrolysis from the 5' end by the T7 exonuclease and then released the short-cleaved FAM-labeled oligonucleotides. Then, the increased FAM signal (FFAM) and the stable CD signal (FCDs) would be tested through a ratiometric routine for the quantification of miRNA-21. The FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05-10 nM, and the detection limit for miRNA-21 was 1 pM with excellent selectivity and reproducibility. Furthermore, this sensor successfully evaluated the expression level of miRNA-21 in clinical blood samples from healthy individuals and gastrointestinal cancer patients, and the results were highly consistent with those of qRT-PCR, suggesting the great clinical application value in the diagnosis of cancer associated with miRNA-21 expression levels.

A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli.

The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology.

Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control.

An elongation cycle of a transcribing RNA polymerase (RNAP) usually consists of multiple kinetics steps, so there exist multiple kinetic checkpoints where non-cognate nucleotides can be selected against. We conducted comprehensive free energy calculations on various nucleotide insertions for viral T7 RNAP employing all-atom molecular dynamics simulations. By comparing insertion free energy profiles between the non-cognate nucleotide species (rGTP and dATP) and a cognate one (rATP), we obtained selection free energetics from the nucleotide pre-insertion to the insertion checkpoints, and further inferred the selection energetics down to the catalytic stage. We find that the insertion of base mismatch rGTP proceeds mainly through an off-path along which both pre-insertion screening and insertion inhibition play significant roles. In comparison, the selection against dATP is found to go through an off-path pre-insertion screening along with an on-path insertion inhibition. Interestingly, we notice that two magnesium ions switch roles of leave and stay during the dATP on-path insertion. Finally, we infer that substantial selection energetic is still required to catalytically inhibit the mismatched rGTP to achieve an elongation error rate ∼10-4 or lower; while no catalytic selection seems to be further needed against dATP to obtain an error rate ∼10-2.

Structures and operating principles of the replisome.

Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo-electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A ß hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates.

We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.

Single-Molecule FRET Analysis of Replicative Helicases.

Over the recent years single-molecule fluorescence resonance energy transfer (smFRET) technique has proven to be one of the most powerful tools for revealing mechanistic insights into helicase activities. Here we describe details of single-molecule FRET assays for probing DNA unwinding activities as well as functional dynamics by replicative helicases in real time. The ability of smFRET to measure the behavior of biomolecules at a nanometer scale enabled us to address how the leading and lagging strand synthesis are coordinated during DNA replication, to resolve DNA unwinding steps of Bacteriophage T7 helicase, and to observe heterogeneous unwinding patterns modulated by the DNA binding domain of E1 helicase. These single-molecule FRET assays are generally applicable to other replicative and nonreplicative hexameric helicases.

The antiviral protein Viperin suppresses T7 promoter dependent RNA synthesis-possible implications for its antiviral activity.

Viperin is a multifunctional interferon-inducible broad-spectrum antiviral protein. Viperin belongs to the S-Adenosylmethionine (SAM) superfamily of enzymes known to catalyze a wide variety of radical-mediated reactions. However, the exact mechanism by which viperin exerts its functions is still unclear. Interestingly, for many RNA viruses viperin was shown to inhibit viral RNA accumulation by interacting with different viral non-structural proteins. Here, we show that viperin inhibits RNA synthesis by bacteriophage T7 polymerase in mammalian cells. This inhibition is specific and occurs at the RNA level. Viperin expression significantly reduced T7-mediated cytoplasmic RNA levels. The data showing that viperin inhibits the bacteriophage T7 polymerase supports the conservation of viperin's antiviral activity between species. These results highlight the possibility that viperin might utilize a broader mechanism of inhibition. Accordingly, our results suggest a novel mechanism involving polymerase inhibition and provides a tractable system for future mechanistic studies of viperin.

Optimal numbers of residues in linkers of DNA polymerase I, T7 primase and DNA polymerase IV.

DNA polymerase I (PolI), T7 primase and DNA polymerase IV (Dpo4) have a common feature in their structures that the two main domains are connected by an unstructured polypeptide linker. To perform their specific enzymatic activities, the enzymes are required to rearrange the position and orientation of one domain relative to the other into an active mode. Here, we show that the three enzymes share the same mechanism of the transition from the inert to active modes and use the minimum numbers of residues in their linkers to achieve the most efficient transitions. The transition time to the finally active mode is sensitively dependent on the stretched length of the linker in the finally active mode while is insensitive to the position and orientation in the initially inert state. Moreover, we find that for any enzyme whose two domains are connected by an unstructured flexible linker, the stretched length (L) of the linker in the finally active mode and the optimal number (Nopt) of the residues in the linker satisfy relation L ≈ αNopt, with α = 0.24-0.27 nm being a constant insensitive to the system.

Synthesis and evaluation of an alkyne-modified ATP analog for enzymatic incorporation into RNA.

Alkyne-modified nucleoside analogs are useful for nucleic acid localization as well as functional and structural studies because of their ability to participate in copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions. Here we describe the synthesis of the triphosphate of 7-ethynyl-8-aza-7-deazaadenosine (7-EAATP) and the enzymatic incorporation of 7-EAA into RNA. The free nucleoside of 7-EAA is taken up by HeLa cells and incorporated into cellular RNA by endogenous RNA polymerases. In addition, 7-EAATP is a substrate for both T7 RNA polymerase and poly (A) polymerase from Escherichia coli in vitro, albeit at lower efficiencies than with ATP. This work adds to the toolbox of nucleoside analogs useful for RNA labeling.

A T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection.

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA-CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity.

Size matters, so does shape: Inhibition of transcription of T7 RNA polymerase by iron(II) clathrochelates.

Coordination and organoelement compounds are rarely proposed as the drug candidates despite their vast potential in the area owing to their strictly controlled geometry and rather extensive surface. This is the first example of the inhibition of transcription in the system of T7 RNA polymerase by cage metal complexes. Their IC50 values reach as low as the nanomolar range, placing them among the most potent metal-based transcription inhibitors.

The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities.

BACKGROUND: The Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts. RESULTS: To determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis. CONCLUSIONS: The results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.

Spermine attenuates the action of the DNA intercalator, actinomycin D, on DNA binding and the inhibition of transcription and DNA replication.

The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion.

Zinc-binding domain of the bacteriophage T7 DNA primase modulates binding to the DNA template.

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.

Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase.

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA(350)/dT(25). On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.

Spontaneous DNA lesions modulate DNA structural transitions occurring at nuclease hypersensitive element III(1) of the human c-myc proto-oncogene.

G quadruplex (G4) DNA is a noncanonical four-stranded DNA structure that can form in G repeats by stacking of planar arrays of four hydrogen-bonded guanines called G quartets, in the presence of potassium ions. In addition to a presumed function in the regulation of gene expression, G4 DNA also localizes to regions often characterized by genomic instability. This suggests that formation of this structure may interfere with DNA transactions, including processing of DNA damage at these sites. Here we have studied the effect of two spontaneous DNA lesions, the abasic site and 8-oxoguanine, on the transition from duplex to quadruplex DNA structure occurring at nuclease hypersensitive element III(1) (NHEIII(1)) of the human c-myc promoter. We show by dimethyl sulfate footprinting and RNA polymerase arrest assays that at physiological concentrations of potassium ions NHEIII(1) folds into two coexisting G4 DNA structures, myc-1245 and myc-2345, depending on which G runs are utilized for G quartet formation. We found that a single substitution of G12 of NHEIII(1) with a single abasic site or a single 8-oxoguanine prevented formation of G4 structure myc-2345 in favor of structure myc-1245, where the lesion was accommodated in a DNA loop formed by G11-AP12/(or 8-oxoG12)-G13-G14. Surprisingly, when an additional G to A base substitution was introduced at position 3 of NHEIII(1), we observed formation of myc-2345. The extent of this structural transition was modulated by the location and type of lesion within the G11-G14 repeat. Our data indicate that spontaneous lesions formed in the G4-forming sequence of c-myc NHEIII(1) affect the structural transitions occurring at this regulatory site, potentially altering transcription factor binding and DNA repair of lesions formed in this highly regulated sequence.

The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7.

DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.

Stepwise motion of a microcantilever driven by the hydrolysis of viral ATPases.

The biomolecular machines involved in DNA packaging by viruses generate one of the highest mechanical powers observed in nature. One component of the DNA packaging machinery, called the terminase, has been proposed as the molecular motor that converts chemical energy from ATP hydrolysis into mechanical movement of DNA during bacteriophage morphogenesis. However, the conformational changes involved in this energy conversion have never been observed. Here we report a real-time measurement of ATP-induced conformational changes in the terminase of bacteriophage T7 (gp19). The recording of the cantilever bending during its functionalization shows the existence of a gp19 monolayer arrangement confirmed by atomic force microscopy of the immobilized proteins. The ATP hydrolysis of the gp19 terminase generates a stepped motion of the cantilever and points to a mechanical cooperative effect among gp19 oligomers. Furthermore, the effect of ATP can be counteracted by non-hydrolyzable nucleotide analogs.