Faculty Bibliography

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.

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.

Anti-CRISPR proteins (Acrs) targeting CRISPR-Cas9 systems represent natural "off switches" for Cas9-based applications. Recently, AcrIIC1, AcrIIC2, and AcrIIC3 proteins were found to inhibit Neisseria meningitidis Cas9 (NmeCas9) activity in bacterial and human cells. Here we report biochemical and structural data that suggest molecular mechanisms of AcrIIC2- and AcrIIC3-mediated Cas9 inhibition. AcrIIC2 dimer interacts with the bridge helix of Cas9, interferes with RNA binding, and prevents DNA loading into Cas9. AcrIIC3 blocks the DNA loading step through binding to a non-conserved surface of the HNH domain of Cas9. AcrIIC3 also forms additional interactions with the REC lobe of Cas9 and induces the dimerization of the AcrIIC3-Cas9 complex. While AcrIIC2 targets Cas9 orthologs from different subtypes, albeit with different efficiency, AcrIIC3 specifically inhibits NmeCas9. Structure-guided changes in NmeCas9 orthologs convert them into anti-CRISPR-sensitive proteins. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms and guidelines for designing regulatory tools in Cas9-based applications.

Although many eukaryotic transcripts contain cap structures, it has been long thought that bacterial RNAs do not carry any special modifications on their 5'-ends. In bacteria, primary transcripts are produced by transcription initiated with a nucleoside triphosphate and are therefore triphosphorylated on 5'-ends. Some transcripts are then processed by nucleases that yield monophosphorylated RNAs for specific cellular activities. Many primary transcripts are also converted to monophosphorylated species by removal of the terminal pyrophosphate for 5'-end-dependent degradation. Recent studies surprisingly revealed an expanded repertoire of chemical groups on 5'-ends of bacterial RNAs. In addition to mono- and triphosphorylated moieties, some mRNAs and sRNAs contain cap-like structures and diphosphates on their 5'-ends. Although incorporation and removal of these groups have become better understood in recent years, the physiological significance of these modifications remain obscure. This review highlights recent studies aimed at identification and elucidation of novel modifications on the 5'-ends of bacterial RNAs and discusses possible physiological applications of the modified RNAs. This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Processing > Capping and 5' End Modifications.

The ykkC family of bacterial riboswitches combines several widespread classes that have similar secondary structures and consensus motifs but control different genes in response to different cellular metabolites. Here we report the crystal structures of two distinct ykkC riboswitches specifically bound to their cognate ligand ppGpp, a second messenger involved in stress response, or PRPP, a precursor in purine biosynthesis. Both RNAs adopt similar structures and contain a conserved core previously observed in the guanidine-specific ykkC riboswitch. However, ppGpp and PRPP riboswitches uniquely employ an additional helical element that joins the ends of the ligand-sensing domains and creates a tunnel for direct and Mg²⁺-mediated binding of ligands. Mutational and footprinting experiments highlight the importance of conserved nucleotides forming the tunnel and long-distance contacts for ligand binding and genetic response. Our work provides new insights into the specificity of riboswitches and gives a unique opportunity for future studies of RNA evolution.

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5' termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5'-end-dependent mRNA degradation begins with the generation of monophosphorylated 5' termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH-DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

Deprotection of the 5' end appears to be a universal mechanism for triggering the degradation of mRNA in bacteria and eukaryotes. In Escherichia coli, for example, converting the 5' triphosphate of primary transcripts to a monophosphate accelerates cleavage at internal sites by the endonuclease RNase E. Previous studies have shown that the RNA pyrophosphohydrolase RppH catalyzes this transformation in vitro and generates monophosphorylated decay intermediates in vivo. Recently, we reported that purified E. coli RppH unexpectedly reacts faster with diphosphorylated than with triphosphorylated substrates. By using a novel assay, it was also determined that diphosphorylated mRNA decay intermediates are abundant in wild-type E. coli and that their fractional level increases to almost 100% for representative mRNAs in mutant cells lacking RppH. These findings indicate that the conversion of triphosphorylated to monophosphorylated RNA in E. coli is a stepwise process involving sequential phosphate removal and the transient formation of a diphosphorylated intermediate. The latter RNA phosphorylation state, which was previously unknown in bacteria, now appears to define the preferred biological substrates of E. coli RppH. The enzyme responsible for generating it remains to be identified.

RNA modifications that once escaped detection are now thought to be pivotal for governing RNA lifetimes in both prokaryotes and eukaryotes. For example, converting the 5'-terminal triphosphate of bacterial transcripts to a monophosphate triggers 5' end-dependent degradation by RNase E. However, the existence of diphosphorylated RNA in bacteria has never been reported, and no biological role for such a modification has ever been proposed. By using a novel assay, we show here for representative Escherichia coli mRNAs that approximately 35%-50% of each transcript is diphosphorylated. The remainder is primarily monophosphorylated, with surprisingly little triphosphorylated RNA evident. Furthermore, diphosphorylated RNA is the preferred substrate of the RNA pyrophosphohydrolase RppH, whose biological function was previously assumed to be pyrophosphate removal from triphosphorylated transcripts. We conclude that triphosphate-to-monophosphate conversion to induce 5' end-dependent RNA degradation is a two-step process in E. coli involving gamma-phosphate removal by an unidentified enzyme to enable subsequent beta-phosphate removal by RppH.

RNA molecules participate in virtually all cellular processes ranging from transfer of hereditary information to gene expression control. In cells, many RNAs form specific interactions with proteins often using short nucleotide sequences for protein recognition. Biochemical and structural studies of such RNA-protein complexes demand preparation of short RNAs. Although short RNAs can be synthesized chemically, certain proteins require monophosphate or triphosphate moieties on the 5' end of RNA. Given high cost of chemical triphosphorylation, broad application of such RNAs is impractical. In vitro transcription of RNA by DNA-dependent bacteriophage T7 RNA polymerase provides an alternative option to prepare short RNAs with different phosphorylation states as well as modifications on the 5' terminus. Here we outline the in vitro transcription methodology employed to prepare