The RNA Polymerase of Marine Cyanophage Syn5

Zhu, Bin; Tabor, Stanley; Raytcheva, Desislava; Hernandez, Alfredo J.; King, Jonathan; Richardson, Charles C.

doi:10.1074/jbc.m112.442350

Cited by 25 publications

(25 citation statements)

References 32 publications

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“…The product of the Syn5 RNA polymerase reaction was confirmed to be RNA by its resistance to DNase I and sensitivity to RNase I (Figure 2B). Addition of KCl increases the yield of transcripts of Syn5 RNA polymerase (Figure 2C), with the highest yield observed at 160 mM KCl, consistent with our previous report (18). However, this stimulation is only observed when circular plasmids are used as templates (described below).…”

Section: Resultssupporting

confidence: 92%

Syn5 RNA polymerase synthesizes precise run-off RNA products

Zhu¹,

Tabor²,

Richardson³

2013

Nucleic Acids Research

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The enzyme predominantly used for in vitro run-off RNA synthesis is bacteriophage T7 RNA polymerase. T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3′-terminus (N + 1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30 000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3′-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3′-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.

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Section: Resultssupporting

confidence: 92%

Syn5 RNA polymerase synthesizes precise run-off RNA products

Zhu¹,

Tabor²,

Richardson³

2013

Nucleic Acids Research

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“…T7 RNAP does not recognize the host’s promoters, and vice versa, the host’s RNAP does not recognize the T7 promoters (Temme et al, 2012 ). Further, it is conceivable that marine cyanophages like Syn5 (Zhu et al, 2013 ), which differ from T7 RNAP in among other things a greater salt tolerance, could fill the same role as an orthogonal RNAP. To conclude, it is our expectation that the development of a broad range of widely applicable cyanobacterial genetic parts will help to enable the use of cyanobacteria as large-scale green producers of the renewable products of the future.…”

Section: Outlook and Suggestions For Future Developmentmentioning

confidence: 99%

Engineered Transcriptional Systems for Cyanobacterial Biotechnology

Camsund

Lindblad

2014

Front. Bioeng. Biotechnol.

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Cyanobacteria can function as solar-driven biofactories thanks to their ability to perform photosynthesis and the ease with which they are genetically modified. In this review, we discuss transcriptional parts and promoters available for engineering cyanobacteria. First, we go through special cyanobacterial characteristics that may impact engineering, including the unusual cyanobacterial RNA polymerase, sigma factors and promoter types, mRNA stability, circadian rhythm, and gene dosage effects. Then, we continue with discussing component characteristics that are desirable for synthetic biology approaches, including decoupling, modularity, and orthogonality. We then summarize and discuss the latest promoters for use in cyanobacteria regarding characteristics such as regulation, strength, and dynamic range and suggest potential uses. Finally, we provide an outlook and suggest future developments that would advance the field and accelerate the use of cyanobacteria for renewable biotechnology.

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“…We have modified the original expression vector ( 15 , 16 ) to improve the expression of His-tagged Syn5 RNA polymerase by removing the internal Syn5 promoter sequence within the Syn5 RNA polymerase gene without changing the encoded amino acid (Supplementary Figure S1A). With this vector (Syn5 RNAP-NP-pET24) the synthesized Syn5 RNA polymerase will not initiate transcription from the internal promoter sequence, an event that would deplete the rNTP pools and inhibit the synthesis of the full-length mRNA for Syn5 RNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

“…Recently we characterized a single-subunit RNA polymerase from marine cyanophage Syn5 ( 15 ) and described some of its advantages as a tool for in vitro transcription. These advantages include product-3′-homogeneity, high processivity, flexible initiating nucleotide and tolerance to salt ( 16 ).…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

et al. 2015

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The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2′-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA polymerase have been identified.

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The RNA Polymerase of Marine Cyanophage Syn5

Cited by 25 publications

References 32 publications

Syn5 RNA polymerase synthesizes precise run-off RNA products

Syn5 RNA polymerase synthesizes precise run-off RNA products

Engineered Transcriptional Systems for Cyanobacterial Biotechnology

Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

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