Syn5 RNA polymerase synthesizes precise run-off RNA products

Abstract

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.

Figure 1.

RNA polymerases used in this study. (A) SDS-PAGE gel of Syn5 RNA polymerase purified as previously reported (Syn5 I, 18), newly purified Syn5 RNA polymerase using Ni-NTA chromatography in the presence of 2 M NaCl (Syn5 IIA), newly purified Syn5 RNA polymerase purified using Ni-NTA and gel filtration chromatography in the presence of 2 M NaCl (Syn5 IIB) and T7 RNA polymerase. (B) Incorporation of AMP at 24°C by 50 nM of each of the RNA polymerases shown in (A). A plasmid with both a Syn5 and T7 promoter was used as the template; each RNA polymerases uses its cognate promoter exclusively to initiate transcription.

Figure 2.

Apparent processivity of Syn5 and T7 RNA polymerases. (A) Agarose gel showing RNA synthesis by previously purified Syn5 RNA polymerase (I), newly purified Syn5 RNA polymerase using the improved procedure (IIB) and T7 RNA polymerase on a plasmid template containing a single Syn5 or T7 promoter, as indicated in the schematics at the left. (B) The product shown in (A) synthesized by Syn5 RNA polymerase IIB was incubated with 1 U/µl RNase I or DNase I to confirm that it contained RNA. (C) Effect of increasing KCl concentration on transcription by Syn5 RNA polymerase. (D) Products synthesized by Syn5 and T7 RNAP in a rolling-circle reaction were analysed under denaturing conditions. (E) Same assay as in (D) except that the plasmid templates were linearized as shown in the schematic. (F) Run-off RNA synthesis by Syn5 and T7 RNA polymerase on template containing a T7 terminator sequence (see schematic). (G) The effect of decreasing concentration of T7 and Syn5 RNA polymerase on the processivity of transcription. (H) RNA polymerase promoter distribution on coliphage T7 and cyanophage Syn5 genomes.

Figure 3.

Comparison of T7 and Syn5 RNA polymerase in run-off synthesis of small RNA. (A) Effect of temperature and salt concentration on RNA synthesis by T7 and Syn5 RNA polymerase. The region of the predicted product and the N + 1 product is enlarged from the region in the black rectangle from the whole gel. (B) The same experiment as described in (A) except that His-tagged T7 RNA polymerase was used.

Figure 4.

Synthesis of tRNA^Arg by Syn5 and T7 RNA polymerase. (A) Synthesis of tRNA^Arg on an analytical scale using varying amounts of Syn5 and T7 RNA polymerase. Reactions were carried out in the presence of radioactively labeled CTP, and either 22, 67 or 200 nM of either Syn5 or T7 RNA polymerase at 37°C. Bands corresponding to the tRNA^Arg and N + 1 products are indicated on the right. (B) Synthesis of tRNA^Arg on a preparative scale using Syn5 and T7 RNA polymerase. The denaturing gel shows an analysis of an aliquot of total RNA produced by Syn5 and T7 RNAP at 37°C in a 200 µl reaction. RNAs were purified by either ZYMO RNA clean kit or phenol/chloroform extraction followed by ethanol precipitation (P/C/E), as noted on top of the gel. The marker (M) in the left lane contains native tRNA^Arg (containing modified bases) that had been overexpressed and purified from E. coli cells. The two major bands in the total RNAs are tRNA^Arg and the N + 1 product as marked. The inset graph shows the charging of arginine on tRNA^Arg from 0.2 µM total RNAs (purified native tRNA^Arg, Syn5 RNA polymerase products or T7 RNA polymerase products) by E. coli total aminoacyl-tRNA synthetases as a function of time. The maximum charging capacity at 30 min was converted into the amount of functional tRNA^Arg molecules (arginine and tRNA^Arg are at a 1:1 ratio in the aminoacylation reaction). Based on this conversion, the percentage of functional tRNA^Arg molecules in the total RNA transcripts in each reaction was calculated and shown in the column chart at the bottom right.

Figure 5.

Synthesis of long run-off products and the incorporation of modified nucleosides by Syn5 and T7 RNA polymerases. (A) The plasmid template shown in the schematic was linearized with the restriction enzyme NotI. Transcripts produced by 10 nM of either Syn5 or T7 RNA polymerase were analysed on an agarose gel. Lane 1 shows the products of reactions in the presence of 160 mM KCl. Lane 2 shows the products of the same reactions carried out in the absence of KCl. Lanes 3 and 4 show the products of reactions where CTP was replaced by 5mCTP (lane 3) and UTP by Ps-UTP (lane 4) in the absence of KCl. (B) Incorporation of UTP or Ps-UTP into small RNA by 22, 67 and 200 nM Syn5 and T7 RNA polymerase as analysed by denaturing TBE PAGE. The region of the gel where full-length run-off products and N + 1 products migrate is shown.

Figure 6.

Preference of initiating nucleotides by Syn5 RNA polymerase. Various DNA templates (as shown schematically) directing the synthesis of transcripts differ by only one initiating nucleotide at the first or second position were transcribed by Syn5 RNA polymerase. The transcripts were separated on a 10% TBE-urea gel and the amount of a 37 nt run-off RNA product (arrow) was quantified and compared as shown in the chart.

Figure 7.

Stability of Syn5 RNA polymerase during prolonged incubations. Syn5 or T7 RNA polymerase reaction was carried out at 37°C. After 1, 2 and 4 h their products were separated on a 15% TBE-urea gel and the run-off tRNA transcripts were shown as white bands by ethidium bromide staining.