NusG-dependent RNA polymerase pausing is a common feature of riboswitch regulatory mechanisms.

Transcription by RNA polymerase is punctuated by transient pausing events. Pausing provides time for RNA folding and binding of regulatory factors to the paused elongation complex. We previously identified 1600 NusG-dependent pauses throughout the Bacillus subtilis genome, with ∼20% localized to 5' leader regions, suggesting a regulatory role for these pauses.

Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in .

RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of grown in the presence and absence of amino acids.

mRNA structural elements immediately upstream of the start codon dictate dependence upon eIF4A helicase activity.

Background: The RNA helicase eIF4A1 is a key component of the translation initiation machinery and is required for the translation of many pro-oncogenic mRNAs. There is increasing interest in targeting eIF4A1 therapeutically in cancer, thus understanding how this protein leads to the selective re-programming of the translational landscape is critical. While it is known that eIF4A1-dependent mRNAs frequently have long GC-rich 5'UTRs, the details of how 5'UTR structure is resculptured by eIF4A1 to enhance the translation of specific mRNAs are unknown.

In Vivo Genome-Wide RNA Structure Probing with Structure-seq.

In vivo genome-wide RNA structure probing provides a global view of RNA structure as it occurs in the cell and can assist in elucidating important functional aspects of RNA structure. Structure-seq2 provides high-quality data on transcriptome-wide RNA structure in vivo but contains numerous steps that require technical precision. In this chapter we present the steps needed to produce high-quality structural data with a focus on controls and troubleshooting.

Genome-wide RNA structurome reprogramming by acute heat shock globally regulates mRNA abundance.

The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown.

StructureFold2: Bringing chemical probing data into the computational fold of RNA structural analysis.

The secondary structure of an RNA is often implicit to its function. Recently, various high-throughput RNA structure probing techniques have been developed to elucidate important RNA structure-function relationships genome-wide. These techniques produce unwieldy experimental data sets that require evaluation with unique computational pipelines.

Glyoxals as in vivo RNA structural probes of guanine base-pairing.

Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines.

Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo.

RNA serves many functions in biology such as splicing, temperature sensing, and innate immunity. These functions are often determined by the structure of RNA. There is thus a pressing need to understand RNA structure and how it changes during diverse biological processes both in vivo and genome-wide.

Eliminating blurry bands in gels with a simple cost-effective repair to the gel cassette.

Gel electrophoresis and subsequent imaging using phosphorimagers is one of the most important and widely used techniques in RNA and DNA analysis. Radiolabeling nucleic acids with P and detecting bands using a phoshorimager are useful both in a qualitative sense for nucleic acid detection and in a quantitative sense for structural, kinetic, or binding-based assays. Because of this, good resolution of gel bands based on molecular weight and size of RNA or DNA is essential for analysis.

Genome-Wide Analysis of RNA Secondary Structure.

Single-stranded RNA molecules fold into extraordinarily complicated secondary and tertiary structures as a result of intramolecular base pairing. In vivo, these RNA structures are not static. Instead, they are remodeled in response to changes in the prevailing physicochemical environment of the cell and as a result of intermolecular base pairing and interactions with RNA-binding proteins.