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RNA template-directed RNA synthesis by T7 RNA polymerase
1Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Summary
Bacteriophage T7 RNA polymerase unexpectedly synthesized longer RNA transcripts. This occurred due to RNA template-directed RNA synthesis, where the initial RNA product acted as a template for further RNA extension.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Synthesis
Background:
- Bacteriophage T7 RNA polymerase is a widely used enzyme for in vitro RNA synthesis.
- Run-off transcription typically terminates at a defined DNA sequence.
- Unexpectedly long transcripts were observed during oligoribonucleotide synthesis.
Purpose of the Study:
- To investigate the mechanism behind the production of unexpectedly long RNA transcripts during T7 RNA polymerase-mediated synthesis.
- To determine if RNA can serve as a template for RNA synthesis using T7 RNA polymerase.
Main Methods:
- DNA-directed run-off transcription using bacteriophage T7 RNA polymerase.
- Analysis of RNA products to identify the synthesis mechanism.
- Investigating RNA template-directed RNA synthesis with various oligoribonucleotides.
Main Results:
- A major transcript significantly longer than expected was produced.
- The extended product resulted from an initial DNA-directed transcription followed by RNA template-directed RNA synthesis.
- RNA template-directed RNA synthesis occurred efficiently, even with marginally stable RNA templates, leading to products more than twice the original length.
Conclusions:
- Bacteriophage T7 RNA polymerase can perform RNA template-directed RNA synthesis.
- Self-complementarity at the 3' end of RNA can create a primed template for further RNA synthesis.
- This mechanism may explain the efficient replication of certain RNAs by T7 RNA polymerase.