Monitoring RNA dynamics in native transcriptional complexes

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)–dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

Metabolite trafficking enables programmed terpene secretion by yeast

Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells 1. We investigated whether engineered microbes could exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner 2,3. In this study, we demonstrated a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking becomes programmable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically coupled a terpene-specific protein with an export signal peptide and subsequently demonstrated exceptionally efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). Other carrier proteins could also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the first and most efficient cognate pathway for metabolite secretion by microorganisms.

Discovery and characterization of a fourth class of guanidine riboswitches

Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.

Thermodynamic constraints are sufficient for the emergence of flux sensors in metabolism

Metabolism is a precisely coordinated phenomenon, the apparent goal of which is to balance fluxes to maintain robust growth. However, coordinating fluxes requires information about rates, which is not obviously reconcilable with known regulatory mechanisms in which concentrations are sensed through metabolite binding. While flux sensor examples have been characterized, the fundamental principles underlying the phenomenon in general are not well understood. Specifically, the questions of which fluxes can be sensed, and the mechanism by which they are remain open. We address this by showing that the concentrations of substrates of thermodynamically constrained reactions reflect upstream flux and therefore carry information about rates which can be propagated through regulatory interactions to control other fluxes in the network. Using fluxomic, metabolomic, and thermodynamic data in E coli, we show that the concentrations of a few metabolites in central carbon metabolism reflect their producing fluxes and demonstrate that they can transmit information about these rates because of their positions in the network and their roles as effectors.

Do the P1 and P2 hairpins of the Guanidine-II riboswitch interact?

Riboswitches regulate genes by adopting different structures in responds to metabolite binding. The guanidine-II riboswitch is the smallest representative of the ykkC class with the mechanism of its function being centred on the idea that its two stem loops P1 and P2 form a kissing hairpin interaction upon binding of guanidinium (Gdm+). This mechanism is based on in-line probing experiments with the full-length riboswitch and crystal structures of the truncated stem loops P1 and P2. However, the crystal structures reveal only the formation of the homodimers P1 | P1 and P2 | P2 but not of the proposed heterodimer P1 | P2. Here, site-directed spin labeling (SDSL) in combination with Pulsed Electron–Electron Double Resonance (PELDOR or DEER) is used to study their structures in solution and how they change upon binding of Gdm+. It is found that both hairpins adopt different structures in solution and that binding of Gdm+ does indeed lead to the formation of the heterodimer but alongside the homodimers in a statistical 1:2:1 fashion. These results do thus support the proposed switching mechanism.

Efficient computation of co-transcriptional RNA-ligand interaction dynamics

Riboswitches form an abundant class of cis-regulatory RNA elements that mediate gene expression by binding a small metabolite. For synthetic biology applications, they are becoming cheap and accessible systems for selectively triggering transcription or translation of downstream genes. Many riboswitches are kinetically controlled, hence knowledge of their co-transcriptional mechanisms is essential. We present here an efficient implementation for analyzing co-transcriptional RNA-ligand interaction dynamics. This approach allows for the first time to model concentration-dependent metabolite binding/unbinding kinetics. We exemplify this novel approach by means of the recently studied I-A 2’-deoxyguanosine (2’dG)-sensing riboswitch from Mesoplasma florum.

High-affinity low-capacity and low-affinity high-capacity N-acetyl-2-aminofluorene (AAF) macromolecular binding sites are revealed during the growth cycle of adult rat hepatocytes in primary culture

ABSTRACTLong-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites – a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II) – associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4 x 10−6 M and BMAX[APPARENT] ≈ 6 pmols/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5 x 10−3 M and BMAX[APPARENT] ≈ 350 pmols/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.