SHAPE-enabled fragment-based ligand discovery for RNA
SignificanceRNA molecules encode proteins and play numerous regulatory roles in cells. Targeting RNA with small molecules, as is routine with proteins, would create broad opportunities for modulating biology and creating new drugs. However, this opportunity has been difficult to realize because creating novel small molecules that bind RNA, especially using modest resources, is challenging. This study integrates two widely used technologies, SHAPE chemical probing of RNA and fragment-based ligand discovery, to craft an innovative strategy for creating small molecules that bind to and modulate the activity of a structured RNA. The anticipated impact is high because the methods are simple, can be implemented in diverse research and discovery contexts, and lead to realistic druglike molecules.
The Non-Coding RNA Journal Club: Highlights on Recent Papers-11 [Editorial]
We are delighted to share with you our eleventh Journal Club and highlight some of the most interesting papers published recently [...].
Subsite Ligand Recognition and Cooperativity in the TPP Riboswitch: Implications for Fragment-Linking in RNA Ligand Discovery
RNA molecules can show high levels of cooperativity in their global folding and interactions with divalent ions. However, cooperativity at individual ligand-RNA interaction sites remains poorly understood. Here, we investigated the binding of thiamine and methylene diphosphonic acid (MDP, a soluble structural analogue of pyrophosphate) to the thiamine pyrophosphate riboswitch. These ligands each bind weakly at proximal subsites, with 10 Î¼M and 1 mM affinities, respectively. The affinity of MDP moderately improves when thiamine or thiamine-like fragments are pre-bound to the RNA. Covalent linking of thiamine and MDP substantially increases riboswitch binding to a notable high affinity of 20 nM. Crystal structures and single-molecule correlated chemical probing revealed favorable induced fit effects upon binding of individual ligands and, unexpectedly, a substantial thermodynamically unfavorable RNA structural rearrangement upon binding of the linked thiamine-MDP ligand. Thus, linking of two ligands of modest affinity, accompanied by an unfavorable structural rearrangement, still yields a potent linked RNA-binding compound. Since complex ligands often bind riboswitches and other RNAs at proximal subsites, principles derived from this work inform and support fragment-linking strategies for identifying small molecules that interact with RNA specifically and with high affinity.
A distinct RNA recognition mechanism governs Np4 decapping by RppH
Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np4) RNA caps. Removal of this cap is critical for initiating 5' end-dependent degradation of those RNAs, potentially affecting bacterial adaptability to stress; however, the predominant Np4 decapping enzyme in proteobacteria, ApaH, is inactivated by the very conditions of disulfide stress that enable Np4-capped RNAs to accumulate to high levels. Here, we show that, in Escherichia coli cells experiencing such stress, the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts, preferring them as substrates over their triphosphorylated and diphosphorylated counterparts. Unexpectedly, this enzyme recognizes Np4-capped 5' ends by a mechanism distinct from the one it uses to recognize other 5' termini, resulting in a one-nucleotide shift in substrate specificity. The unique manner in which capped substrates of this kind bind to the active site of RppH positions the Î´-phosphate, rather than the Î²-phosphate, for hydrolytic attack, generating triphosphorylated RNA as the primary product of decapping. Consequently, a second RppH-catalyzed deprotection step is required to produce the monophosphorylated 5' terminus needed to stimulate rapid RNA decay. The unconventional manner in which RppH recognizes Np4-capped 5' ends and its differential impact on the rates at which such termini are deprotected as a prelude to RNA degradation could have major consequences for reprogramming gene expression during disulfide stress.
Growing a garden of fluorescent RNAs
Riboswitch Mechanisms: New Tricks for an Old Dog
Discovered almost twenty years ago, riboswitches turned out to be one of the most common regulatory systems in bacteria, with representatives found in eukaryotes and archaea. Unlike many other regulatory elements, riboswitches are entirely composed of RNA and capable of modulating expression of genes by direct binding of small cellular molecules. While bacterial riboswitches had been initially thought to control production of enzymes and transporters associated with small organic molecules via feedback regulatory circuits, later findings identified riboswitches directing expression of a wide range of genes and responding to various classes of molecules, including ions, signaling molecules, and others. The 5'-untranslated mRNA regions host a vast majority of riboswitches, which modulate transcription or translation of downstream genes through conformational rearrangements in the ligand-sensing domains and adjacent expression-controlling platforms. Over years, the repertoire of regulatory mechanisms employed by riboswitches has greatly expanded; most recent studies have highlighted the importance of alternative mechanisms, such as RNA degradation, for the riboswitch-mediated genetic circuits. This review discusses the plethora of bacterial riboswitch mechanisms and illustrates how riboswitches utilize different features and approaches to elicit various regulatory responses.
Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance
Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine Î³-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.
Cooperativity and Allostery in RNA Systems
Allostery is among the most basic biological principles employed by biological macromolecules to achieve a biologically active state in response to chemical cues. Although initially used to describe the impact of small molecules on the conformation and activity of protein enzymes, the definition of this term has been significantly broadened to describe long-range conformational change of macromolecules in response to small or large effectors. Such a broad definition could be applied to RNA molecules, which do not typically serve as protein-free cellular enzymes but fold and form macromolecular assemblies with the help of various ligand molecules, including ions and proteins. Ligand-induced allosteric changes in RNA molecules are often accompanied by cooperative interactions between RNA and its ligand, thus streamlining the folding and assembly pathways. This chapter provides an overview of the interplay between cooperativity and allostery in RNA systems and outlines methods to study these two biological principles.
Principles of RNA and nucleotide discrimination by the RNA processing enzyme RppH
All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the Escherichia coli Nudix hydrolase RppH, which triggers 5'-end-dependent RNA degradation by removing orthophosphate from the 5'-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5'-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5'-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5'-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.
T-box RNA gets boxed