Syn5 RNA polymerase synthesizes precise run-off RNA products

Bin Zhu,Charles C Richardson,Stanley Tabor

doi:10.1093/nar/gkt1193

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.

Highlights

RNA plays fundamental roles in cell physiology and is an important target for biomedical research and biotechnology
RNA transcripts synthesized by RNA polymerase in vitro are used widely in applications that include hybridization analysis, NMR and crystallographic structural studies, biochemical and genetic studies, and the preparation of functional molecules such as tRNA, mRNA, sRNA, ribozymes and aptamers
The other two enzymes available for run-off RNA synthesis are bacteriophage T3 and SP6 RNA polymerase [1,6,7,8], which have properties similar to those of T7 RNA polymerase. Problems encountered with these RNA polymerases include limited processivity, high salt sensitivity [9], undesired products resulting from abortive synthesis [10] and most significantly, the addition of a non-base-paired nucleotide at the 30 end of the run-off transcript [4,6]