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AssigneeThe University of Chicago
InventorsKrystyna M. Kazmierczak, Elena K. Davydova, Lucia B. Rothman-Denes
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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.
An isolated nucleic acid comprising a region encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:15.
An isolated polynucleotide comprising a sequence identical or completely complementary to the full length of SEQ ID NO:1.
An isolated polynucleotide comprising a sequence identical or completely complementary to the full length of SEQ ID NO:3.
An isolated nucleic acid encoding the polypeptide of SEQ ID NO:4 wherein said polypeptide has a mutation at position Y678.
A method of making a full-length N4 vRNAP or mini-vRNAP comprising:
Dependencies are inferred from the claim text.