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.
Kinetic Mechanism of GTP Binding and RNA Synthesis during Transcription Initiation by Bacteriophage T7 RNA Polymerase
