Mutants Of T7 RNA Polymerase Research Articles

Enzymatic Synthesis of Chimeric DNA Oligonucleotides by in Vitro Transcription with dTTP, dCTP, dATP, and 2'-Fluoro Modified dGTP.

Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates.

Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides.

Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.

T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA–DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.

Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.

On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2′-O-methyl modified RNA with yields much higher than enzymes currently employed.

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host's native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.

The synthesis of 2′-methylseleno adenosine and guanosine 5′-triphosphates

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2′-SeCH3-uridine and 2′-SeCH3-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2′-methylseleno-2′-deoxyadenosine and -guanosine 5′-triphosphates (2′-SeCH3-ATP and 2′-SeCH3-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.

Enzymatic synthesis of 2′-methylseleno-modified RNA

Selenium-derivatization of RNA is a powerful and advantageous alternative to conventional heavy atom derivatization techniques that are required for the phasing of X-ray crystallographic diffraction data. Among several possibilities, the 2′-methylseleno (2′-SeCH3) modification has been most widely explored and was responsible for a series of important RNA structure determinations, such as the Diels–Alder ribozyme or complexes of antibiotics to HIV dimerization initiation site (DIS) RNA. So far, 2′-SeCH3-RNA has only been accessible by chemical solid-phase synthesis for sizes of up to 50 nucleotides and up to about 100 nucleotides in combination with enzymatic ligation procedures. To overcome this limitation, here we present the enzymatic synthesis of 2′-SeCH3-RNA to open up access for the preparation of long selenium-modified RNA sequences, which cannot be accomplished by conventional chemical synthesis. Therefore, we first elaborated a synthetic route towards the 2′-methylseleno-2′-deoxyribonucleoside triphosphates of cytosine and uridine (2′-SeCH3–CTP and 2′-SeCH3–UTP). With these crucial derivatives in hand, we found that mutants of T7 RNA polymerase are able to incorporate 2′-SeCH3–CMP and 2′-SeCH3–UMP into RNA, while the wild-type polymerase fails to do so. This study demonstrates the efficient enzymatic synthesis of 2′-SeCH3-modified RNA and, thus, provides a thorough foundation for an alternative derivatization strategy in X-ray crystallographic structure analysis of larger RNAs. Such efforts are currently highly requested because of the steadily increasing number of novel non-coding RNAs whose structural features remain to be elucidated.

Evidence for novel processing of the anaerobically inducible dicistronic focA‐pfl mRNA transcript in Escherichia coli

The anaerobically inducible dicistronic focA-pfl operon is transcribed from three co-ordinately regulated promoters that are located 5' of the operon. Remarkably, the 5' ends of four further highly abundant operon-internal transcripts are located within the focA gene, with a fifth transcript mapping in the intergenic region between focA and pfl. The findings of this study demonstrate that the bulk of these five operon-internal transcripts are the result of processing. Processing was independent of the broad-spectrum endoribonucleases associated with mRNA turnover and still occurred when the upstream regulatory region of the operon was replaced with two different heterologous promoters recognized by Escherichia coli core RNA polymerase, including the tetP promoter. However, when the T7Phi10 promoter was introduced upstream of the focA-pfl operon, mainly full-length transcript and a minor amount of two processing products were observed. T7 RNA polymerase mutants that exhibit reduced elongation speed did not restore the wild-type transcript-processing pattern. Moreover, processing was independent of focA translation. Taken together, these data suggest that processing of the focA-pfl transcripts occurs by a novel mechanism that might require the action of E. coli core RNA polymerase.

Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase.

Bacteriophage T7 RNA polymerase is a single-subunit enzyme which has a C-terminal amino acid sequence of Phe-Ala-Phe-Ala883 (FAFA883). Closely related hydrophobic sequences are present at the C termini of seven other single-subunit RNA polymerases, including the mitochondrial RNA polymerase. Mutations at any of the four C-terminal residues depress initiation rates of T7 RNA polymerase from 50 to 95%, accompanied by large increases in the K(m) values for the initiating nucleotide, GTP, as well as the K(m)'s for promoter DNA. The dramatic drops in initiation rates shown by the mutant enzymes remain after correcting for any alteration in saturation of the enzyme by the initiating nucleotide or the promoter DNA resulting from the changes in K(m). In contrast, the high processivity of the enzyme is not altered by mutations in the last four residues. However, the propensity for the enzyme to add an untemplated nucleotide at the 3'-ends of transcripts is abolished by the A880AFA883 mutation. The C-terminal FAFA sequence or foot appears to interact both with the initiating NTP and with the most downstream nucleotides of the promoter, possibly through hydrophobic interactions with the minor groove, in the region where free radical footprinting of the polymerase-promoter DNA complex suggests that the enzyme binds across the minor groove.

Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA

A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and S641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution S641A loses RNA polymerase activity but is able to synthesize DNA.

Isolation of Transcriptionally Active Mutants of T7 RNA Polymerase that do not Support Phage Growth

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8– 9appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time. f2 f2 Abbreviations used: IPTG, isopropyl-β- d-thiogalactopyranoside; aa, amino acid(s).

Characterization of a set of T7 RNA polymerase active site mutants.

We have evaluated the elongation rates, processivities, and abortive transcription characteristics of a set of T7 RNA polymerase mutants that map to the polymerase active site. The effects of these mutations on transcription are complex: they cause decreases in activity and processivity during both the processive and abortive phases of transcription and exhibit disproportionate decreases in activity and processivity on poly(dA).poly(dT) or poly(dT) versus poly(dG).poly(dC) templates. They also exhibit an increase in the proportion of slippage dependent poly(G) transcript synthesis during the initial stages of transcription. It is shown that these multiple, distinct effects on transcription can be attributed to decreases in the mutant enzymes' phosphodiester bond formation rates. Estimates of the decreases in these rates are derived from the measured transcript elongation rates and processivities of the mutant enzymes.

T7 RNA polymerase mutants with altered promoter specificities.

The amino acid at position 748 in T7 RNA polymerase (RNAP) functions to discriminate base pairs at positions -10 and -11 in the promoter. We have constructed a series of T7 RNAP mutants having all possible amino acid substitutions at this position. Surprisingly, most (13/19) substitutions result in active RNAPs, and many of these exhibit altered promoter specificities. Identification of mutant RNAPs with altered specificities expands the repertoire of highly specific phage RNAPs that are available for use in phage RNAP-based transcription systems and highlights the complexity of sequence-specific DNA recognition.

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase.

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.

Isolation and characterization of mutant bacteriophage T7 RNA polymerases

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.