Project description:An alternative sigma factor (σ32) recognizes the unique set of promoters upon heat shock. Here, we determined 54 σ32 promoters at nucleotide resolution using ChIP-exo, enabling us to compare those with housekeeping σ70 promoters. The results elucidated the overarching principles of promoter overlapping between the two σ-factors, which are sequence-specific non-, half-, and full-shared modes with a perfect sequence conservativeness of −35 element as a key determinant of full-shared mode.
Project description:An alternative sigma factor (σ32) recognizes the unique set of promoters upon heat shock. Here, we determined 54 σ32 promoters at nucleotide resolution using ChIP-exo, enabling us to compare those with housekeeping σ70 promoters. The results elucidated the overarching principles of promoter overlapping between the two σ-factors, which are sequence-specific non-, half-, and full-shared modes with a perfect sequence conservativeness of −35 element as a key determinant of full-shared mode.
Project description:Here, we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to conduct a target-specific and genome-wide profile of in vivo RNA secondary structure in rice (Oryza sativa). Our study presents an optimized DMS-MaPseq for probing in vivo RNA structure in rice.
Project description:Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.
Project description:Although DNA motifs recognized by the transcription factors (TFs) have been determined, challenges remain in probing in vivo architecture of TF-DNA complexes on a genome-wide scale. Here, we show in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using chromatin immunoprecipitation coupled with sequencing (ChIP-exo). The identified 62 ArgR-binding loci were classified into three groups, comprised of single, double, and triple peak-pairs, respectively. Each peak-pair has unique 93 bp-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequence. Moreover, the peak-pairs provided the three ArgR-binding modes defined by the position of the two ARG boxes, indicating that the formation of DNA bending apparently centered between the pair of ARG boxes facilitates the non-specific contacts between ArgR subunits and the residual sequences. Thus, our data postulate the in vivo architecture of ArgR-DNA complexes to understand its transcription regulatory mechanism. ChIP-exo profiles of ArgR (+Arginine) and ArgR (-Arginine) were generated by deep sequencing in duplicates using Illumina MiSeq.