SMARTT: C. tetani glycine riboswitch (type-1) transcribed with 25 mM ... - SRA

SRX4232645: SMARTT: C. tetani glycine riboswitch (type-1) transcribed with 25 mM Gly - Replicate A
1 ILLUMINA (Illumina HiSeq 4000) run: 17.1M spots, 5.2G bases, 2Gb downloads

Design: A mutagenized library of template DNA was made by error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies). Mutations were introduced to the aptamer and expression platform domains using a 16 cycle error-prone PCR reaction. Nonmutagenized regions upstream of the aptamer, including a promoter, were added through a two-step PCR reaction with Phusion HF polymerase (NEB or Thermo Fisher Scientific). This library was used as DNA template for an in vitro transcription reaction containing the specified concentration of ligand (glycine or alanine). DNA template was removed by DNase treatment with TURBO DNase (Thermo Fisher Scientific). A pre-adenylated adaptor was then added to the 3' end using T4 RNA Ligase II, Trunc. K227Q (NEB). Using this adaptor as a handle, the RNA was reverse-transcribed into DNA with SuperScript III (Thermo Fisher Scientific). Illumina adaptors were then added through a two-step PCR reaction with Phusion HF polymerase (NEB or Thermo Fisher Scientific).

Submitted by: Yale University

Study: Gene regulation by a glycine riboswitch singlet utilizes a finely tuned energetic landscape for helical switching

PRJNA476609 • SRP150789 • All experiments • All runs

show Abstracthide Abstract

Riboswitches contain structured aptamer domains that, upon ligand-binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. The sequencing data provided here were generated using SMART, a high-throughput technique for monitoring in vitro transcription termination. This dataset reveals the functional effects of all 522 single point mutants and several combinations of double mutants of the glycine riboswitch type-1 singlet from Clostridium tetani. These data highlight functionally relevant regions of the RNA and describe the energetic contributions ligand binding provides to the expression platform.

Sample: Cte type-1 singlet - SMART with 25 mM Gly

SAMN09445541 • SRS3431284 • All experiments • All runs

Organism: Clostridium tetani E88

Library:

Name: Cte-10

Instrument: Illumina HiSeq 4000

Strategy: RNA-Seq

Source: OTHER

Selection: RT-PCR

Layout: PAIRED

Spot descriptor:

1 forward151 reverse

Runs: 1 run, 17.1M spots, 5.2G bases, 2Gb

Run	# of Spots	# of Bases	Size	Published
SRR7359563	17,090,538	5.2G	2Gb	2018-07-21

ID:: 5719651

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