A histidine-tagged, deletion mutant of bacteriophage N4-coded, virion RNA
polymerase (mini-vRNAP) which is active has been developed. The
his-tagged mini-vRNAP has been cloned under the control of the pBAD
promoter, is stable and is purified in a single step yielding large
amounts (10 mg/liter of E. coli expressing cells). This RNA polymerase
uses single-stranded DNA containing 17 bases (the promoter) upstream of
the transcribed regions as a template. In the presence of E. coli SSB
protein, it transcribes this template efficiently, providing a unique
system to synthesize RNAs of the desired sequence using single-stranded
DNA templates. The enzyme incorporates derivatized nucleoside
triphosphates with high efficiency. A mutant of mini-vRNAP has been
generated that incorporates deoxynucleoside triphosphates. In addition,
the inventors have developed an in vivo system to express RNAs and
proteins under mini vRNA polymerase promoter control.