RNA Chaperone Protein Research Articles

Molecular Changes Associated With Tumor Initiation and Progression of Soft Tissue Sarcomas: Targeting the Genome and Epigenome.

Soft tissue sarcomas are rare, but generally aggressive tumors which disproportionately affect children and young adults. They represent less than 10% of all cancers, but are one of the most frequently diagnosed cancers in pediatric patients. These cancers have a high rate of morbidity and mortality, and their overall incidence has been increasing at an estimated rate of 26% over the last 2 decades. The cause of this increased incidence is unknown but various environmental factors have been implicated. Establishing standard therapeutic strategies is challenging for soft tissue sarcomas as more than 50 different histological subtypes exist, each with their own molecular alterations and clinical characteristics, and this combination of tumor heterogeneity and a limited number of clinical cases make detailed omics level molecular studies particularly challenging. This chapter will focus on the unique genetic and epigenetic changes which characterize these cancers, with an emphasis on translocation-associated sarcomas involving primary gene fusions with the RNA chaperone protein EWSR1. We will highlight current therapeutic approaches and discuss opportunities for targeted molecular therapeutics.

Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803.

Cyanobacteria are an attractive host for biofuel production because they can produce valuable chemical compounds from CO2 fixed by photosynthesis. However, the available genetic tools that enable precise gene regulation for the applications of synthetic biology are insufficient. Previously, we engineered an RNA-based posttranscriptional regulator, termed riboregulator, for the control of target gene expression in cyanobacterium Synechocystis sp. PCC 6803. Moreover, we enhanced the gene regulation ability of the riboregulators in Escherichia coli by fusing and engineering a scaffold sequence derived from naturally occurring E. coli noncoding small RNAs. Here, we demonstrated that the scaffold sequence fused to the riboregulators improved their gene regulation ability in Synechocystis sp. PCC 6803. To further improve gene regulation, we expressed an exogenous RNA chaperone protein that is responsible for noncoding small RNA-mediated gene regulation, which resulted in higher target gene expression. The scaffold sequence derived from natural E. coli noncoding small RNAs is effective for designing RNA-based genetic tools and scaffold-fused riboregulators are a strong RNA-tool to regulate gene expression in cyanobacteria.

New insight into the structure and function of Hfq C-terminus.

Accumulating evidence indicates that RNA metabolism components assemble into supramolecular cellular structures to mediate functional compartmentalization within the cytoplasmic membrane of the bacterial cell. This cellular compartmentalization could play important roles in the processes of RNA degradation and maturation. These components include Hfq, the RNA chaperone protein, which is involved in the post-transcriptional control of protein synthesis mainly by the virtue of its interactions with several small regulatory ncRNAs (sRNA). The Escherichia coli Hfq is structurally organized into two domains. An N-terminal domain that folds as strongly bent β-sheets within individual protomers to assemble into a typical toroidal hexameric ring. A C-terminal flexible domain that encompasses approximately one-third of the protein seems intrinsically unstructured. RNA-binding function of Hfq mainly lies within its N-terminal core, whereas the function of the flexible domain remains controversial and largely unknown. In the present study, we demonstrate that the Hfq-C-terminal region (CTR) has an intrinsic property to self-assemble into long amyloid-like fibrillar structures in vitro. We show that normal localization of Hfq within membrane-associated coiled structures in vivo requires this C-terminal domain. This finding establishes for the first time a function for the hitherto puzzling CTR, with a plausible central role in RNA transactions.

The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae

Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilm matrix protein (RbmC) by base-pairing with the 5′ region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA down-regulates Vrp protein synthesis by base-pairing to the 5′ region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.

Wait and capture: unwinding the strategy of a DEAD-box helicase.

Wait and capture: unwinding the strategy of a DEAD-box helicase.

Vibrio cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein.

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor σE. In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5′ untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the σE-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.

Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.

Hfq reduces envelope stress by controlling expression of envelope‐localized proteins and protein complexes in enteropathogenic Escherichia coli

Gram-negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ(E) envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two-component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram-negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ(E) envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ(E) and Cpx responses in non-pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ(E) response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K-12. Cpx pathway activation resulted in part from overexpression of the bundle-forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.

PZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

BackgroundThe ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications.ResultsA quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli – Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified.ConclusionsWe show that a shuttle vector-based protein affinity ‘pull-down’ approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.

Template Role of Double-Stranded RNA in Tombusvirus Replication

Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.

Improving the Gene-Regulation Ability of Small RNAs by Scaffold Engineering in Escherichia coli

Noncoding small RNAs are involved in transcriptional and post-transcriptional gene regulation of target mRNAs by modulating mRNA elongation, stability, or translational efficiency. Many natural trans-encoded small RNAs contain a scaffold that allows binding of the RNA chaperone protein Hfq for conditional gene regulation. Here, we improved the gene regulation abilities of small RNAs by directly fusing the natural Escherichia coli trans-encoded small RNA-derived scaffolds, including Hfq-binding and rho-independent transcription terminator sequences, to the 3' end of the small RNAs that mediate RNA-based gene regulation. As target small RNAs to improve their gene regulation abilities, we selected small RNAs of artificial post-transcriptional riboregulators and transcriptional attenuators. Four different small RNA scaffolds were fused to the riboregulator and attenuator-derived small RNAs. Mutations were introduced into the best small RNA scaffold to improve its gene-regulation ability further. As a result, mutations predicted to stabilize the secondary structures of the small RNA scaffolds dramatically increased its ability to regulate gene expression of both the post-transcriptional riboregulator and transcriptional attenuator systems. We believe our engineered small RNA scaffolds are applicable to other RNA regulators for improving regulatory activity, and engineered small RNA scaffolds may present a valuable strategy to regulate target gene expression strongly.

The RNA chaperone Hfq regulates expression of fimbrial-related genes and virulence ofSalmonella entericaserovar Enteritidis

Salmonella Enteritidis is an intracellular pathogen that causes enteritis and systemic disease in humans and other animals. The RNA chaperone protein Hfq mediates the binding of small noncoding RNAs to target mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we constructed an hfq deletion mutant in S.Enteritidis SE50336 and analyzed the expression of major fimbrial subunits sefA, bcfA, fimA, safA, stbA, sthA, csgA, csgD, and pegA using quantitative real-time PCR. The gene expression of sefA increased about 14-fold in the hfq mutant, as compared with its expression in the wild-type strain. The expression of fimA and pegA did not change significantly, while the expression of the other fimbrial genes was significantly down-regulated in the hfq mutant. The ability of SE50336Δhfq adhering to Caco-2 cells was also reduced as compared with wild-type adherence. The virulence of the hfq mutant was significantly reduced in a 1-day-old chicken model of S.Enteritidis disease, as determined by quantifying the lethal dose 50% of the bacterial strains. We conclude that Hfq critically contributes to S.Enteritidis virulence, likely partially affected by regulating fimbrial gene expression.

Shewanella oneidensisHfq promotes exponential phase growth, stationary phase culture density, and cell survival

BackgroundHfq is an RNA chaperone protein that has been broadly implicated in sRNA function in bacteria. Here we describe the construction and characterization of a null allele of the gene that encodes the RNA chaperone Hfq in Shewanella oneidensis strain MR-1, a dissimilatory metal reducing bacterium.ResultsLoss of hfq in S. oneidensis results in a variety of mutant phenotypes, all of which are fully complemented by addition of a plasmid-borne copy of the wild type hfq gene. Aerobic cultures of the hfq∆ mutant grow more slowly through exponential phase than wild type cultures, and hfq∆ cultures reach a terminal cell density in stationary phase that is ~2/3 of that observed in wild type cultures. We have observed a similar growth phenotype when the hfq∆ mutant is cultured under anaerobic conditions with fumarate as the terminal electron acceptor, and we have found that the hfq∆ mutant is defective in Cr(VI) reduction. Finally, the hfq∆ mutant exhibits a striking loss of colony forming units in extended stationary phase and is highly sensitive to oxidative stress induced by H2O2 or methyl viologen (paraquat).ConclusionsThe hfq mutant in S. oneidensis exhibits pleiotropic phenotypes, including a defect in metal reduction. Our results also suggest that hfq mutant phenotypes in S. oneidensis may be at least partially due to increased sensitivity to oxidative stress.

Characterization of MicA interactions suggests a potential novel means of gene regulation by small non-coding RNAs

MicA is a small non-coding RNA that regulates ompA mRNA translation in Escherichia coli. MicA has an inhibitory function, base pairing to the translation initiation region of target mRNAs through short sequences of complementarity, blocking their ribosome-binding sites. The MicA structure contains two stem loops, which impede its interaction with target mRNAs, and it is thought that the RNA chaperone protein Hfq, known to be involved in MicA regulation of ompA, may structurally remodel MicA to reveal the ompA-binding site for cognate pairing. To further characterize these interactions, we undertook biochemical and biophysical studies using native MicA and a ‘stabilized’ version, modified to mimic the conformational state of MicA where the ompA-binding site is exposed. Our data corroborate two proposed roles for Hfq: first, to bring both MicA and ompA into close proximity, and second, to restructure MicA to allow exposure of the ompA-binding site for pairing, thereby demonstrating the RNA chaperone function of Hfq. Additionally, at accumulated MicA levels, we identified a Mg2+-dependent self-association that occludes the ompA-recognition region. We discuss the potential contribution of an Mg2+-mediated conformational switch of MicA for the regulation of MicA function.

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

BackgroundUpon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized.ResultsWe examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells.ConclusionsProfiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN.

Construction and characterization of a null allele of the hfq gene of the dissimilatory metal reducing bacterium Shewanella oneidensis

Shewanella oneidensis is a dissimilatory metal reducing bacterium that can utilize a range of terminal electron acceptors when grown under anaerobic conditions. Our primary goal is to identify and characterize genes that encode small regulatory non‐coding RNAs (sRNAs) in S. oneidensis. sRNA genes are regulated by changes in environmental conditions and can themselves regulate other genes by inexact base pairing to their mRNA targets. We are particularly interested in sRNAs that may regulate the process of metal reduction.The highly conserved RNA chaperone protein Hfq has been widely implicated in bacterial sRNA function. We have constructed a null allele of the S. oneidensis hfq gene. The S. oneidensis hfq mutant has a modest delayed growth phenotype. In addition, our mutant displays reduced survival in stationary phase. Our hfq mutant also exhibits a decreased rate of Cr(VI) reduction when grown under anaerobic conditions compared to the rate of Cr(VI) reduction by wild type S. oneidensis. We are currently evaluating the growth and survival of the Shewanella hfq null mutant. We are also investigating the mutant's sensitivity to a variety of physical stresses, evaluating the capacity of the hfq mutant to reduce metals other than Cr(VI), and determining the role of Shewanella hfq in sRNA function.This research is funded by a grant from the RI‐INBRE program, award # P20RR016457‐10.

Tracking Bacterial Riboregulation by DsrA Noncoding RNA

Small regulatory noncoding RNAs (sRNA) are evolutionarily conserved molecules that act by base pairing with mRNAs in response to environmental stress. They can affect cell physiology in numerous ways, including changes in cell morphology, remodeling the bacterial cell surface and decreasing growth rates. These stress-related sRNAs require the RNA chaperone protein Hfq for their regulatory function. Most of the sRNAs, for which a mechanistic understanding is available, act as inhibitors of translation by base pairing with their mRNA target around the ribosome-binding site (rbs). Nevertheless, there are also some examples, as in the case of DsrA, where the sRNA plays a role of both a translational inhibitor of the bacterial actin-like MreB, and a translational activator by exposing the mRNA rbs and promoting ribosome binding to rpoS mRNA (encoding the sigma S transcriptional factor). We recently observed that the E. coli DsrA sRNA is present in few copies in the bacterial cell. For this reason, the ability of the sRNA to bind an mRNA and regulate protein translation can be viewed essentially as a single-molecule process in individual cells, probably contributing to variation from cell to cell in a population. Our aim is to analyze the dynamics of DsrA-dependent translation in response to various stresses at the single molecule level using single-molecule FRET assays. Dye-labeled synthetic RNA fragments of DsrA and of its targets were used to follow and quantify Hfq-dependent RNA annealing. The speed and efficiency of annealing are directly influenced by physico-chemical parameters, such as temperature, and changes in sRNA concentration encountered during physiological stresses.

The Azoarcus Group I Intron Ribozyme Misfolds and Is Accelerated for Refolding by ATP-dependent RNA Chaperone Proteins

Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg(2+) concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding.

ProQ Is an RNA Chaperone that Controls ProP Levels in Escherichia coli

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.