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Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase
Author(s) -
Gudima S.O,
Kostyuk D.A,
Grishchenko O.I,
Tunitskaya V.L,
Memelova L.V,
Kochetkov S.N
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01393-3
Subject(s) - t7 rna polymerase , microbiology and biotechnology , transcription (linguistics) , rna polymerase , chemistry , mutant , polynucleotide , plasmid , polymerase , promoter , rna , oligonucleotide , nucleotide , biology , dna , biochemistry , gene , escherichia coli , bacteriophage , gene expression , linguistics , philosophy
Synthesis of deoxynucleotide‐containing RNA‐like single‐stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter‐containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8–10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1–+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non‐coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG‐containing) was achieved with high yields.