Faculty Bibliography

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Conformational rearrangements are key to the function of riboswitches. These regulatory mRNA regions specifically bind to cellular metabolites using evolutionarily conserved sensing domains and modulate gene expression via adjacent downstream expression platforms, which carry gene expression signals. The regulation is achieved through the ligand-dependent formation of two alternative and mutually exclusive conformations involving the same RNA region. While X-ray crystallography cannot visualize dynamics of such dramatic conformational rearrangements, this method is pivotal to understand RNA-ligand interaction that stabilize the sensing domain and drive folding of the expression platform. X-ray crystallography can reveal local changes in RNA necessary for discriminating cognate and noncognate ligands. This chapter describes preparation of thiamine pyrophosphate riboswitch RNAs and its crystallization with different ligands, resulting in structures with local conformational changes in RNA. These structures can help to derive information on the dynamics of the RNA essential for specific binding to small molecules, with potential for using this information for developing designer riboswitch-ligand systems.

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.

We are delighted to share with you our eleventh Journal Club and highlight some of the most interesting papers published recently [...].

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 μM 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.

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 δ-phosphate, rather than the β-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.

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.

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 γ-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.