Transcription Increases the Cooperativity of Ribonucleoprotein Assembly.

The synthesis of new ribosomes begins during transcription of the rRNA and is widely assumed to follow an orderly 5' to 3' gradient. To visualize co-transcriptional assembly of ribosomal protein-RNA complexes in real time, we developed a single-molecule platform that simultaneously monitors transcription and protein association with the elongating transcript. Unexpectedly, the early assembly protein uS4 binds newly made pre-16S rRNA only transiently, likely due to non-native folding of the rRNA during transcription. Stable uS4 binding became more probable only in the presence of additional ribosomal proteins that bind upstream and downstream of protein uS4 by allowing productive assembly intermediates to form earlier. We propose that dynamic sampling of elongating RNA by multiple proteins overcomes heterogeneous RNA folding, preventing assembly bottlenecks and initiating assembly within the transcription time window. This may be a common feature of transcription-coupled RNP assembly.

Small RNAs and Hfq capture unfolded RNA target sites during transcription.

Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq⋅DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq⋅DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfq⋅DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.

Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing.

Bacterial small RNAs (sRNAs) regulate the expression of hundreds of transcripts via base pairing mediated by the Hfq chaperone protein. sRNAs and the mRNA sites they target are heterogeneous in sequence, length, and secondary structure. To understand how Hfq can flexibly match diverse sRNA and mRNA pairs, we developed a single-molecule Förster resonance energy transfer (smFRET) platform that visualizes the target search on timescales relevant in cells. Here we show that unfolding of target secondary structure on Hfq creates a kinetic energy barrier that determines whether target recognition succeeds or aborts before a stable anti-sense complex is achieved. Premature dissociation of the sRNA can be alleviated by strong RNA-Hfq interactions, explaining why sRNAs have different target recognition profiles. We propose that the diverse sequences and structures of Hfq substrates create an additional layer of information that tunes the efficiency and selectivity of non-coding RNA regulation in bacteria.

Cooperative tertiary interaction network guides RNA folding.

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends on the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.

Co-transcriptional folding of the glmS ribozyme enables a rapid response to metabolite.

The glmS ribozyme riboswitch, located in the 5' untranslated region of the Bacillus subtilis glmS messenger RNA (mRNA), regulates cell wall biosynthesis through ligand-induced self-cleavage and decay of the glmS mRNA. Although self-cleavage of the refolded glmS ribozyme has been studied extensively, it is not known how early the ribozyme folds and self-cleaves during transcription. Here, we combine single-molecule fluorescence with kinetic modeling to show that self-cleavage can occur during transcription before the ribozyme is fully synthesized. Moreover, co-transcriptional folding of the RNA at a physiological elongation rate allows the ribozyme catalytic core to react without the downstream peripheral stability domain. Dimethyl sulfate footprinting further revealed how slow sequential folding favors formation of the native core structure through fraying of misfolded helices and nucleation of a native pseudoknot. Ribozyme self-cleavage at an early stage of transcription may benefit glmS regulation in B. subtilis, as it exposes the mRNA to exoribonuclease before translation of the open reading frame can begin. Our results emphasize the importance of co-transcriptional folding of RNA tertiary structure for cis-regulation of mRNA stability.

A roadmap for rRNA folding and assembly during transcription.

Ribonucleoprotein (RNP) assembly typically begins during transcription when folding of the newly synthesized RNA is coupled with the recruitment of RNA-binding proteins (RBPs). Upon binding, the proteins induce structural rearrangements in the RNA that are crucial for the next steps of assembly. Focusing primarily on bacterial ribosome assembly, we discuss recent work showing that early RNA-protein interactions are more dynamic than previously supposed, and remain so, until sufficient proteins are recruited to each transcript to consolidate an entire domain of the RNP. We also review studies showing that stable assembly of an RNP competes against modification and processing of the RNA. Finally, we discuss how transcription sets the timeline for competing and cooperative RNA-RBP interactions that determine the fate of the nascent RNA. How this dance is coordinated is the focus of this review.

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfq's integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry coupled with surface-induced dissociation and molecular dynamics simulations to disentangle the arrangement of the CTDs and their impact on the stability of Escherichia coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding.

Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.

Small ribozymes such as Oryza sativa twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg2+ and Mn2+ ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

RbfA and IF3 couple ribosome biogenesis and translation initiation to increase stress tolerance.

Bacterial ribosome biogenesis and translation occur in the same cellular compartment. Therefore, a biochemical gate-keeping step is required to prevent error-prone immature ribosomes from engaging in protein synthesis. Here, we provide evidence for a previously unknown quality control mechanism in which the abundant ribosome assembly factor, RbfA, suppresses protein synthesis by immature Escherichia coli 30S subunits. After 30S maturation, RbfA is displaced by initiation factor 3 (IF3), which promotes translation initiation. Genetic interactions between RbfA and IF3 show that RbfA release by IF3 is important during logarithmic growth as well as during stress encountered during stationary phase, low nutrition, low temperature, and antibiotics. By gating the transition from 30S biogenesis to translation initiation, RbfA and IF3 maintain the fidelity of bacterial protein synthesis.

Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation.

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.

In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates.

Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E. coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription in vivo. Quantitative (14)N/(15)N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly.

Role of Era in assembly and homeostasis of the ribosomal small subunit.

Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

Protein-guided RNA dynamics during early ribosome assembly.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

A metastable rRNA junction essential for bacterial 30S biogenesis.

Tertiary sequence motifs encode interactions between RNA helices that create the three-dimensional structures of ribosomal subunits. A Right Angle motif at the junction between 16S helices 5 and 6 (J5/6) is universally conserved amongst small subunit rRNAs and forms a stable right angle in minimal RNAs. J5/6 does not form a right angle in the mature ribosome, suggesting that this motif encodes a metastable structure needed for ribosome biogenesis. In this study, J5/6 mutations block 30S ribosome assembly and 16S maturation in Escherichia coli. Folding assays and in-cell X-ray footprinting showed that J5/6 mutations favor an assembly intermediate of the 16S 5' domain and prevent formation of the central pseudoknot. Quantitative mass spectrometry revealed that mutant pre-30S ribosomes lack protein uS12 and are depleted in proteins uS5 and uS2. Together, these results show that impaired folding of the J5/6 right angle prevents the establishment of inter-domain interactions, resulting in global collapse of the 30S structure observed in electron micrographs of mutant pre-30S ribosomes. We propose that the J5/6 motif is part of a spine of RNA helices that switch conformation at distinct stages of assembly, linking peripheral domains with the 30S active site to ensure the integrity of 30S biogenesis.

Metals induce transient folding and activation of the twister ribozyme.

Twister is a small ribozyme present in almost all kingdoms of life that rapidly self-cleaves in variety of divalent metal ions. We used activity assays, bulk FRET and single-molecule FRET (smFRET) to understand how different metal ions promote folding and self-cleavage of the Oryza sativa twister ribozyme. Although most ribozymes require additional Mg2+ for catalysis, twister inverts this expectation, requiring 20-30 times less Mg2+ to self-cleave than to fold. Transition metals such as Co2+, Ni2+ and Zn2+ activate twister more efficiently than Mg2+ ions. Although twister is fully active in ≤ 0.5 mM MgCl2, smFRET experiments showed that the ribozyme visits the folded state infrequently under these conditions. Comparison of folding and self-cleavage rates indicates that most folding events lead to catalysis, which correlates with metal bond strength. Thus, the robust activity of twister reports on transient metal ion binding under physiological conditions.

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA.

The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq's RNA chaperone activity, the function of Hfq's intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA⋅mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA-mRNA interactions and rapidly cycle between competing targets in the cell.

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, P. aeruginosa transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation of antR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation of antR, demonstrating that the PrrF sRNAs interact with the antR 5' untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. The P. aeruginosa Hfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd [dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to the antR mRNA (0.3 µM), and PrrF was able to bind to antR mRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to the antR UTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, in P. aeruginosaIMPORTANCE The iron-responsive PrrF sRNAs play a central role in regulating P. aeruginosa iron homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and the antR mRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target in P. aeruginosa.

Mimicking Co-Transcriptional RNA Folding Using a Superhelicase.

Vectorial folding of RNA during transcription can produce intermediates with distinct biochemical activities. Here, we design an artificial minimal system to mimic cotranscriptional RNA folding in vitro. In this system, a presynthesized RNA molecule begins to fold from its 5'-end, as it is released from a heteroduplex by an engineered helicase that translocates on the complementary DNA strand in the 3'-to-5' direction. This chemically stabilized "superhelicase" Rep-X processively unwinds thousands of base pairs of DNA. The presynthesized RNA enables us to flexibly position fluorescent labels on the RNA for single-molecule fluorescence resonance energy transfer analysis and allows us to study real-time conformational dynamics during the vectorial folding process. We observed distinct signatures of the maiden secondary and tertiary folding of the Oryza sativa twister ribozyme. The maiden vectorial tertiary folding transitions occurred faster than Mg2+-induced refolding, but were also more prone to misfolding, likely due to sequential formation of alternative secondary structures. This novel assay can be applied to studying other kinetically controlled processes, such as riboswitch control and RNA-protein assembly.

Entropic stabilization of folded RNA in crowded solutions measured by SAXS.

Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cell's interior. Large crowder molecules such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg2+ concentrations typical of the cell's interior. To understand the thermodynamic origins of stabilization by crowder molecules, small angle X-ray scattering was used to measure the folding and helix assembly of a bacterial group I ribozyme at different temperatures and in different MgCl2 and polyethylene glycol (PEG) concentrations. The resulting phase diagrams show that perturbations to folding by each variable do not overlap. A favorable enthalpy change drives the formation of compact, native-like structures, but requires Mg2+ ions at all temperatures studied (5-55°C). PEG reduces the entropic cost of helix assembly and increases correlations between RNA segments at all temperatures. The phase diagrams also revealed a semi-compact intermediate between the unfolded and folded ensemble that is locally more flexible than the unfolded state, as judged by SHAPE modification. These results suggest that environmental variables such as temperature and solute density will favor different types of RNA structures.