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.
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.
SUMMARY 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 cryo-electron microscopy-guided model of the intact HCV IRES and its interaction with 40S ribosomal subunits.
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.
The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 59 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 29-OMe oligonucleotides.
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.
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.
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 E. 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 interacting with many sRNAs and mRNAs, 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 A-form RNA duplex. INTRODUCTION Regulatory, small RNAs (sRNAs) are found in nearly all bacterial species and implicated in important processes such as virulence, biofilm formation, host interactions and antibiotic resistance.(1-3) These sRNAs typically regulate messenger RNA (mRNA) translation through imperfect base pairing near an mRNA's ribosomal binding site.(2, 4-6) In many bacterial species, the stability and function of sRNAs are supported by global RNA-binding proteins, such as the protein Hfq.(1, 4, 7-9) Given that Hfq is not present in all bacterial species and that not all sRNAs depend on Hfq for their function, there is increasing interest in other RNA-binding proteins that may play a role in global gene-regulation in bacteria,(2, 10-13) including a class of proteins that contain FinO domains.(14-17) The Escherichia coli protein FinO is the founding member of the FinO structural class of RNA-binding proteins. In E. coli, FinO binds the FinP sRNA and regulates the 5´ untranslated region (UTR) of traJ.(18, 19) Similarly, Legionella pneumophila RocC contains a FinO-domain and binds the sRNA RocR along with at least four 5' UTRs of mRNAs involved in competence.(20) In E. coli, another FinO-domain-containing protein called ProQ was initially characterized as an RNA-binding protein contributing to osmoregulation through expression of proP.(21) ProQ was recently identified through Grad-Seq experiments to bind to dozens of cellular RNAs,(17) including a large number of sRNAs and mRNA 3'UTRs in Samonella and E. coli.(22) ProQ binding has been shown to regulate mRNA-expression levels through interactions with both 5' and 3' UTRs. It has been shown to form a ternary complex with an sRNA (RaiZ) and an mRNA (hupA), to support RaiZ's repression of hupA,(23) and to protect mRNAs from exonucleolytic degradation by binding to 3' ends.(22) Further, ProQ supports the sRNA SraL in preventin...
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