T7 RNA polymerase transcription with 5-position modified UTP derivatives.
Seven UTP derivatives modified at the 5-position through an amide linkage were tested as substrates for T7 RNA polymerase (T7 RNAP) transcription. All UTP derivatives gave good yields of full-length transcript even from DNA templates that showed a significant number of abortive transcripts using unmodified UTP. A kinetic assay to determine the relative K(m) and V(max) for T7 RNAP transcription gave surprisingly similar values for UTP and the 5-position hydrophobic modifications phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl. The 5-position modifications imidazole and amino, which could both be positively charged, gave K(m) values significantly higher than UTP. All seven UTP derivatives gave relative V(max) values similar to UTP, indicating that insertion of these modified bases into the transcript did not impede its elongation.
Vaught
Jonathan D
JD
College of Physical and Mathematical Sciences, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Dewey
Torin
T
Eaton
Bruce E
BE
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000502:physiology*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins
D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate