New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III

Transcription
Lodoe LamaKevin Ryan

Abstract

Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize ...Continue Reading

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Citations

Oct 4, 2017·Nucleic Acids Research·James M BurkeChristopher S Sullivan
Aug 28, 2020·Proceedings of the National Academy of Sciences of the United States of America·Aravind RamanathanNayef Jarrous
Feb 25, 2021·Transcription·Nayef Jarrous, Alexander Rouvinski

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Methods Mentioned

BETA
immunoprecipitation
in vitro transcription
DNA
electrophoresis
transfection

Software Mentioned

mfold
ImageQuant
MD
DPAGE

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