Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

Xueling Lu; Hui Wu; Heng Xia; Fengtao Huang; Yan Yan; Bingbing Yu; Rui Cheng; Zuzanna Drulis-Kawa; Bin Zhu

doi:10.3389/fmicb.2019.02487

Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

Front Microbiol. 2019 Oct 31:10:2487. doi: 10.3389/fmicb.2019.02487. eCollection 2019.

Authors

Xueling Lu¹, Hui Wu¹, Heng Xia¹, Fengtao Huang¹, Yan Yan¹, Bingbing Yu¹, Rui Cheng¹, Zuzanna Drulis-Kawa², Bin Zhu¹

Affiliations

¹ Key Laboratory of Molecular Biophysics, the Ministry of Education, College of Life Sciences and Technology and Shenzhen College, Huazhong University of Science and Technology, Wuhan, China.
² Institute of Genetics and Microbiology, University of Wrocław, Wrocław, Poland.

Abstract

We have characterized the single subunit RNA polymerase from Klebsiella phage KP34. The enzyme is unique among known bacteriophage RNA polymerases in that it recognizes two unrelated promoter sequences, which provided clues for the evolution of phage single-subunit RNA polymerases. As the first representative enzyme from the "phiKMV-like viruses" cluster, its use in run-off RNA synthesis was investigated. RNA-Seq analysis revealed that the KP34 RNA polymerase does not possess the undesired self-templated RNA terminus extension known for T7 RNA polymerase and is suitable to synthesize RNAs with structured 3' termini such as sgRNAs. A KP34 RNA polymerase Y603F mutant is engineered to incorporate deoxy- and 2'-fluoro ribonucleotide into RNA.

Keywords: T7 RNA polymerase; in vitro transcription; phiKMV; run-off RNA synthesis; sgRNA.