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Transcription Start Site Mapping Using Super-low Input Carrier-CAGE
Published on: June 26, 2019
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Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage,
Irina O Vvedenskaya1, Yuanchao Zhang2, Seth R Goldman1
1Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA; Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
Molecular Cell
|December 3, 2015
Summary
This study introduces the MASTER technology to map bacterial transcription start sites (TSSomes). MASTER reveals DNA sequences significantly impact RNA transcript yield and identifies DNA topology
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Understanding transcription start site (TSS) selection and regulation is crucial for gene expression control.
- Existing methods have limitations in comprehensively profiling TSS inventories and their regulatory determinants.
- Escherichia coli RNA polymerase serves as a model system for studying fundamental transcription initiation mechanisms.
Purpose of the Study:
- To develop a high-throughput sequencing technology for comprehensive analysis of transcription initiation.
- To define the complete set of transcription start sites (TSSomes) for E. coli RNA polymerase.
- To elucidate DNA sequence and topological determinants governing TSS selection, transcript yield, and slippage synthesis.
Main Methods:
- Development of massively systematic transcript end readout (MASTER) technology for DNA library construction and RNA analysis.
- Application of MASTER to profile in vitro and in vivo TSSomes of E. coli.
- Systematic variation of DNA sequences, DNA topology, and NTP concentrations to assess their impact on transcription.
Main Results:
- MASTER technology enables the construction of DNA libraries with up to ~16,000 barcoded sequences.
- Identified full inventories of transcription start sites (TSSomes) for E. coli RNA polymerase.
- Demonstrated that DNA sequences profoundly affect transcript yield (up to 100-fold) and that slippage synthesis is common.
- Showed that TSSomes are dependent on DNA topology, supporting models involving transcription-bubble dynamics ('scrunching' and 'anti-scrunching').
Conclusions:
- The MASTER technology provides a powerful tool for comprehensive analysis of transcription initiation.
- TSS-region DNA sequences are critical determinants of transcript yield and slippage synthesis.
- DNA topology plays a significant role in TSS selection, likely through influencing transcription-bubble formation and collapse.
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