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SP6 RNA polymerase stutters when initiating from an AAA... sequence.
P R Cunningham1, C J Weitzmann, J Ofengand
1Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Nucleic Acids Research
|September 11, 1991
Summary
SP6 polymerase efficiently transcribes Escherichia coli 16S ribosomal RNA genes, though it sometimes adds extra adenosines due to enzyme
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The 16S ribosomal RNA gene is crucial for bacterial protein synthesis.
- Promoter elements control gene transcription initiation.
- T7 and SP6 polymerases are commonly used RNA polymerases in molecular biology.
Purpose of the Study:
- To investigate the transcriptional efficiency of T7 and SP6 promoters driving the Escherichia coli 16S ribosomal RNA gene.
- To analyze the fidelity and initiation characteristics of SP6 and T7 polymerases.
Main Methods:
- Cloning the 16S rRNA gene under T7 and SP6 promoter control.
- In vitro transcription using T7 and SP6 RNA polymerases.
- Analysis of transcript products by sequencing and quantification.
Main Results:
- Both T7 and SP6 polymerases transcribed the 16S rRNA coding sequence accurately.
- SP6 polymerase showed a five-fold higher initiation efficiency compared to T7 polymerase.
- SP6 polymerase generated transcripts with additional adenosine residues at the 5' end in 40-50% of products, attributed to enzyme 'stuttering'.
Conclusions:
- SP6 polymerase is a highly efficient enzyme for transcribing the 16S rRNA gene.
- SP6 polymerase exhibits 'stuttering' during transcription initiation, leading to non-template-encoded nucleotide addition.
- Understanding these initiation dynamics is important for optimizing in vitro transcription systems.