Generation of single-cysteine E. coli ProQ variants to study RNA-protein interaction mechanisms.

ProQ is a FinO-domain protein found in E. coli and other proteobacteria that has a global RNA-binding profile. In order to probe the detailed mechanism of RNA interactions, we have developed a collection of 13 E. coli ProQ variants that possess single-cysteine residues at varied positions on the surface of the N-terminal FinO domain and retain the ability to bind well to RNA. This set of variant ProQ proteins will support future biochemical and biophysical studies to map the orientation of bound RNAs to different sites around the ProQ protein, shedding light on the mechanism of ProQ-RNA interactions.

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ.

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and noncoding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli, though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQ's FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNA's terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70, and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ.

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and non-coding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli , though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQ's FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNA's terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70 and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.

A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA-protein interactions.

This protocol describes a bacterial three-hybrid (B3H) assay, an in vivo system that reports on RNA-protein interactions and can be implemented in both forward and reverse genetic experiments. The B3H assay connects the strength of an RNA-protein interaction inside of living Escherichia coli cells to the transcription of a reporter gene (here, lacZ). We present protocols to (1) insert RNA and protein sequences into appropriate vectors for B3H experiments, (2) detect putative RNA-protein interactions with both qualitative and quantitative readouts and (3) carry out forward genetic mutagenesis screens. The B3H assay builds on a well-established bacterial two-hybrid system for genetic analyses. As a result, protein-protein interactions can be assessed in tandem with RNA interactions with a bacterial two-hybrid assay to ensure that protein variants maintain their functionality. The B3H system is a powerful complement to traditional biochemical methods for dissecting RNA-protein interaction mechanisms: RNAs and proteins of interest do not need to be purified, and their interactions can be assessed under native conditions inside of a living bacterial cell. Once cloning has been completed, an assay can be completed in under a week and a screen in 1-2 weeks.

Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter protein.

Noncoding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system and will facilitate exploration of the binding mechanisms of these interactions.

Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein.

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3'-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least 2 bp preceding an 3' oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5' side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3' and cspE-3' RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3' intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ.

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo.

The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA-protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein-protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2CP)) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfq mutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA-protein interactions.

Crystal structure of the HCV IRES central domain reveals strategy for start-codon positioning.

Translation of hepatitis C viral proteins requires an internal ribosome entry site (IRES) located in the 5' untranslated region of the viral mRNA. The core domain of the hepatitis C virus (HCV) IRES contains a four-way helical junction that is integrated within a predicted pseudoknot. This domain is required for positioning the mRNA start codon correctly on the 40S ribosomal subunit during translation initiation. Here, we present the crystal structure of this RNA, revealing a complex double-pseudoknot fold that establishes the alignment of two helical elements on either side of the four-helix junction. The conformation of this core domain constrains the open reading frame's orientation for positioning on the 40S ribosomal subunit. This structure, representing the last major domain of HCV-like IRESs to be determined at near-atomic resolution, provides the basis for a comprehensive cryoelectron microscopy-guided model of the intact HCV IRES and its interaction with 40S ribosomal subunits.

Historical review of the commissioning of health care disciplines in the USPHS.

PURPOSE: The purpose of this investigation was to examine the commissioning history of the professions of physicians, dentists, nurses, nurse practitioners, physician assistants and dental hygienists of the United States Public Health Service (USPHS), and to determine a critical pathway to commissioning new health care professions into the USPHS. The Advanced Dental Hygiene Practitioner (ADHP), recently developed by the American Dental Hygienists' Association, is an oral health care provider proposed for public health settings that shares the same goal as the USPHS of treating underserved populations in the U.S. With the establishment of the ADHP role, an opportunity for the ADHP to be commissioned into the USPHS may arise. METHODS: Journal articles, books and Web sites documenting the history and commissioning process of the USPHS were researched. Interviews with key USPHS commissioned officers involved with the commissioning process of the selected health care disciplines were conducted. A qualitative comparative analysis to examine published documents and interpret interviews was performed to reveal patterns of events leading to commissioning. Systematic, time-oriented visual displays of data were constructed to identify critical pathways for commissioning new professions into the USPHS. RESULTS: The need for health care professionals to provide quality health care to the Federal beneficiaries of the USPHS was found to be the driving force behind commissioning the selected health care professions into the USPHS. A critical pathway for commissioning new professions into the USPHS was identified. CONCLUSION: Understanding the commissioning process of new health care professions into the USPHS would assist with defining the critical pathway for future USPHS commissioning of the ADHP.

Optimized high-throughput screen for hepatitis C virus translation inhibitors.

Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The authors developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5' capped mRNA control. The authors screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1700 initial hits. After secondary counterscreening, the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end, the authors found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, as well we the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit.

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5' untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine-Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2'-OMe oligonucleotides.

Diastereoselective intramolecular pinacol couplings of sulfinyl iron(0) diene complexes.

3-(2-Formylaryl)-1-sulfinyl-(1Z,3E)-pentadien-5-al iron tricarbonyl complexes were prepared to examine the feasibility and diastereoselectivity of intramolecular pinacol couplings on such substrates. It was found that the pinacol coupling, promoted by VCl(3) x (THF)(3)/Zn, proceeded in good yield and with high diastereoselectivity (>23:1 dr), provided the 2-formylaryl unit remained unsubstituted at the aryl C3 position (ortho to the formyl group). In these latter cases the pinacol coupling was diastereorandom. A 3-formyl-4-(2-formylaryl)-1-sulfinyl-(1Z,3E)-butadiene iron tricarbonyl complex also underwent diastereoselective pinacol coupling (22:1 dr). 3-(3-Formylindolyl)-1-sulfinyl-(1Z,3E)-pentadien-5-al iron tricarbonyl complexes were also prepared, though pinacol coupling of these substrates proceeded in, at best, modest yield for two of the four examples tested. All cases described herein represent the first intramolecular pinacol couplings performed on the periphery of an iron(0) diene tricarbonyl complex.

Dual-surface-modified bacteriophage MS2 as an ideal scaffold for a viral capsid-based drug delivery system.

With the development of covalent modification strategies for viral capsids comes the ability to convert them into modular carrier systems for drug molecules and imaging agents. With this overall goal in mind, we have used two orthogonal modification strategies to decorate the exterior surface of genome-free MS2 capsids with PEG chains, while installing 50-70 copies of a fluorescent dye inside as a drug cargo mimic. Despite the very high levels of modification, the capsids remained in the assembled state, as determined by TEM, size-exclusion chromatography, and dynamic light scattering analysis. The ability of the polymer coating to block the access of polyclonal antibodies to the capsid surface was probed using a sandwich ELISA, which indicated a 90% reduction in binding. Further experiments indicated that biotin groups placed at the distal ends of the polymer chains were still capable of binding to streptavidin, despite their proximity to the PEG layer. Finally, a modular strategy was developed for the attachment of small-molecule targeting groups to the polymer chains through an efficient oxime formation reaction. As a result of these studies, a robust and versatile new platform has emerged for the potential delivery of therapeutic cargo.

eIF3j is located in the decoding center of the human 40S ribosomal subunit.

Protein synthesis in all cells begins with the ordered binding of the small ribosomal subunit to messenger RNA (mRNA) and transfer RNA (tRNA). In eukaryotes, translation initiation factor 3 (eIF3) is thought to play an essential role in this process by influencing mRNA and tRNA binding through indirect interactions on the backside of the 40S subunit. Here we show by directed hydroxyl radical probing that the human eIF3 subunit eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S subunit, placing eIF3j directly in the ribosomal decoding center. eIF3j also interacts with eIF1A and reduces 40S subunit affinity for mRNA. A high affinity for mRNA is restored upon recruitment of initiator tRNA, even though eIF3j remains in the mRNA-binding cleft in the presence of tRNA. These results suggest that eIF3j functions in part by regulating access of the mRNA-binding cleft in response to initiation factor binding.