RNA Hairpin Research Articles

RNA Polymerase Accommodates a Pause RNA Hairpin by Global Conformational Rearrangements that Prolong Pausing

Sequence-specific pausing by RNA polymerase (RNAP) during transcription plays crucial and diverse roles in gene expression. In bacteria, RNA structures are thought to fold within the RNA exit channel of the RNAP and can increase pause lifetimes significantly. The biophysical mechanism of pausing is uncertain. We used single-particle cryo-EM to determine structures of paused complexes, including a 3.8-Å structure of an RNA hairpin-stabilized, paused RNAP that coordinates RNA folding in the his operon attenuation control region of E.coli. The structures revealed a half-translocated pause state (RNA post-translocated, DNA pre-translocated) that can explain transcriptional pausing and a global conformational change of RNAP that allosterically inhibits trigger loop folding and can explain pause hairpin action. Pause hairpin interactions with the RNAP RNA exit channel suggest how RNAP guides the formation of nascent RNA structures.

Conformational Dynamics of the HIV-1 Trans-Activation Response Element RNA Hairpin Bound to a Lab-Evolved Peptide

We report microsecond-length classical molecular dynamics simulations of a lab-evolved peptide bound to the HIV-1 trans-activation response element (TAR) RNA hairpin. An essential 5′-structure in all HIV-1 mRNAs, TAR interacts with HIV-1 Tat to promote HIV-1 genome transcription, and is considered a promising therapeutic target. Using yeast display, a U1A-derived TAR binding protein (TBP) was selected that binds TAR with subnanomolar affinity and extraordinarily high specificity over the native U1A U1hpII target, thereby preventing Tat-dependent transcription in vitro. A co-crystal structure of TBP in complex with TAR reveals the basis for its unprecedented affinity and specificity. The complex exhibits a Watson-Crick C30-G34 base pair in the six-nucleotide apical loop. From chemical modification experiments and sequence conservation, this base pair has been posited as a prerequisite for Tat binding. We investigated the role of a short TBP peptide - responsible for the preponderance of TAR readout - in stabilizing the observed TAR structure using molecular dynamics simulations of TAR in the apo state and in complex with the full TBP protein and the short TBP peptide. To define discrete conformational states, we performed principal component analysis and clustered trajectories based on the resulting principal components. We observed that the apical loop C30-G34 base pair and a major groove U23-A27-U38 base triple are stable features of peptide-bound TAR on the microsecond timescale. In agreement with NMR studies of free TAR, dissolution of the major groove base triple occurs in trajectories of the apo state. The occupancy of contacts between the protein and the TAR major groove is greater for the arginine residues whose mutation to alanine has a greater impact on the binding affinity, as measured by isothermal calorimetry.

Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests

RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

RNA force field with accuracy comparable to state-of-the-art protein force fields

Molecular dynamics (MD) simulation has become a powerful tool for characterizing at an atomic level of detail the conformational changes undergone by proteins. The application of such simulations to RNA structures, however, has proven more challenging, due in large part to the fact that the physical models ("force fields") available for MD simulations of RNA molecules are substantially less accurate in many respects than those currently available for proteins. Here, we introduce an extensive revision of a widely used RNA force field in which the parameters have been modified, based on quantum mechanical calculations and existing experimental information, to more accurately reflect the fundamental forces that stabilize RNA structures. We evaluate these revised parameters through long-timescale MD simulations of a set of RNA molecules that covers a wide range of structural complexity, including single-stranded RNAs, RNA duplexes, RNA hairpins, and riboswitches. The structural and thermodynamic properties measured in these simulations exhibited dramatically improved agreement with experimentally determined values. Based on the comparisons we performed, this RNA force field appears to achieve a level of accuracy comparable to that of state-of-the-art protein force fields, thus significantly advancing the utility of MD simulation as a tool for elucidating the structural dynamics and function of RNA molecules and RNA-containing biological assemblies.

A 2',2'-disulfide-bridged dinucleotide conformationally locks RNA hairpins.

The synthesis and the impact of a disulfide bridge between 2'-O-positions of two adjacent nucleotides in an RNA duplex and in the loop of RNA hairpins are reported. The incorporation of this 2',2'-disulfide (S-S) bridge enabled thermal and enzymatic stabilization of the hairpin depending on its position in the loop. The influence of the disulfide bridge on RNA folding was studied at the HIV Dimerization Initiation Site (DIS) as an RNA sequence model. We have shown that this S-S bridge locked the hairpin form, whereas the extended duplex form was generated after the reduction of the disulfide bond in the presence of tris(2-carboxyethyl)phosphine or glutathione. Thus, the S-S bridge can be useful for understanding RNA folding; an RNA molecular beacon locked by an S-S bridge was also investigated as a sensor for the detection of glutathione.

RNA Interference Ex Vivo.

RNA interference (RNAi) is a widely used technique to regulate the expression of genes and proteins with a high degree of specificity that is not easily accessed by traditional pharmacological approaches. For preclinical research on rheumatoid arthritis (RA), silencing of target genes in primary immune cells can be easily achieved by application of small interfering RNA (siRNA) and synthetic short hairpin RNA (shRNA). Cellular and systemic administration of siRNA or shRNA has been a significant advance in preclinical research on RA. In this chapter, the basic techniques for gene silencing in human-derived peripheral T cells using liposome-dependent siRNA transfection and lentiviral-mediated shRNA delivery, aiming at gene silencing of therapeutic targets, are introduced.

Design of RNA hairpin modules that predictably tune translation in yeast.

Modular parts for tuning translation are prevalent in prokaryotic synthetic biology but lacking for eukaryotic synthetic biology. Working in Saccharomyces cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5′ untranslated region (5′UTR) of mRNAs can be used to tune expression levels by 100-fold by inhibiting translation. We determine the relationship between the calculated free energy of folding in the 5′UTR and in vivo protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this new tool, computational RNA sequence design can be used to predictably fine-tune protein production for genes expressed in yeast.

Triggering nucleic acid nanostructure assembly by conditional kissing interactions.

Nucleic acids are biomolecules of amazing versatility. Beyond their function for information storage they can be used for building nano-objects. We took advantage of loop–loop or kissing interactions between hairpin building blocks displaying complementary loops for driving the assembly of nucleic acid nano-architectures. It is of interest to make the interaction between elementary units dependent on an external trigger, thus allowing the control of the scaffold formation. To this end we exploited the binding properties of structure-switching aptamers (aptaswitch). Aptaswitches are stem–loop structured oligonucleotides that engage a kissing complex with an RNA hairpin in response to ligand-induced aptaswitch folding. We demonstrated the potential of this approach by conditionally assembling oligonucleotide nanorods in response to the addition of adenosine.

Mirror-image aptamer kissing complex for arginine-vasopressin sensing

The recently reported aptamer kissing complex (AKC) strategy has allowed for the development of a new kind of sandwich-like sensing tools. Currently AKC assays have been only applied to low molecular weight molecules and their functionality in complex matrices remains challenging. The objective of the present study broken down into two sub-aims; exploring the propensity to broaden the scope of detectable analytes and designing a more robust system for potential applications to realistic samples. An all L-configuration aptaswitch module derived from a hairpin spiegelmer specific to a larger target, i.e. the arginine-vasopressin (AVP) hormone, was elaborated. The target-induced AKC formation in presence of a specific mirror-image RNA hairpin (L-aptakiss) probe were analyzed by using fluorescence anisotropy. The mirror-image kissing complex was successfully formed when the L-AVP target bound to the engineered L-aptaswitch element. It was also established that the use of methanol as cosolvent significantly improved the assay sensitivity through the stabilization of the ternary complex. Finally, the capability of the mirror-image method to operate in 10-fold diluted, untreated human serum was illustrated. The current work revealed that the AKC concept can be expanded to a wider range of targets and converted to a L-configuration sensing platform especially suitable for bioanalysis purposes.

Structural features of the interaction of the 3′-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting

We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3′-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1–3 in the HEK293 cell mRNA 3′ UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3′ UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.

The intrinsically disordered N-terminal arm of the brome mosaic virus coat protein specifically recognizes the RNA motif that directs the initiation of viral RNA replication.

In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis. RNA binding causes the peptide to change from heterogeneous structures to a single family of structures. Fluorescence anisotropy, fluorescence quenching and size exclusion chromatography experiments all confirm that the NTA can specific recognize the RNA motif. The peptide introduced into plants along with BMV virion increased accumulation of the BMV CP and accelerated the rate of minus-strand RNA synthesis. The intrinsically disordered BMV NTA could thus specifically recognize BMV RNAs to affect viral infection.

Conformational Plasticity of Hepatitis C Virus Core Protein Enables RNA-Induced Formation of Nucleocapsid-like Particles

Many of the unanswered questions associated with hepatitis C virus assembly are related to the core protein (HCVcp), which forms an oligomeric nucleocapsid encompassing the viral genome. The structural properties of HCVcp have been difficult to quantify, at least in part because it is an intrinsically disordered protein. We have used single-molecule Förster Resonance Energy Transfer techniques to study the conformational dimensions and dynamics of the HCVcp nucleocapsid domain (HCVncd) at various stages during the RNA-induced formation of nucleocapsid-like particles. Our results indicate that HCVncd is a typical intrinsically disordered protein. When it forms small ribonucleoprotein complexes with various RNA hairpins from the 3′ end of the HCV genome, it compacts but remains intrinsically disordered and conformationally dynamic. Above a critical RNA concentration, these ribonucleoprotein complexes rapidly and cooperatively assemble into large nucleocapsid-like particles, wherein the individual HCVncd subunits become substantially more extended.

Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA-DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991-1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

FGF19 genetic amplification as a potential therapeutic target in lung squamous cell carcinomas.

BackgroundAlthough FGF 19 gene aberrations are associated with carcinogenesis and progression in human cancers, the roles of FGF 19 genetic amplification and expression in Chinese patients with lung squamous cell carcinoma (LSCC) and FGF 19 amplification as a potential therapeutic target for LSCC are not well understood.MethodsFluorescence in situ hybridization analysis and quantitative real‐time‐PCR was used to detect FGF 19 genetic amplification and FGF 19 messenger RNA expression in LSCC tumor and paired adjacent samples. Small interfering RNA and short hairpin RNA were used to knockdown FGF19 in vitro and in vivo. Results FGF 19 amplification was identified in a subset of LSCC patients (37.5%, 15/40), and upregulation of FGF 19 expression was found in 60% (24/40) of tumor tissues compared to adjacent non‐tumorous tissues. Correlation analysis with clinicopathologic parameters showed that FGF 19 upregulation was significantly associated with heavy smoking. Small interfering RNA knockdown of FGF 19 led to the significant inhibition of cell growth and induced apoptosis in LSCC cells carrying the amplified FGF 19 gene, but these effects was not observed in non‐amplified LSCC cells. Interfering FGF 19 expression with short hairpin RNA also resulted in tumor growth inhibition and induced apoptosis in LSCC xenografts with amplified FGF 19 in tumor cells.ConclusionOur results suggested that FGF 19 signaling activation is required for cell growth and survival of FGF 19 amplified LSCC cells, both in vitro and in vivo. Intervention of FGF 19 activation could be a potential therapeutic strategy for LSCC patients with FGF 19 amplification.

Abstract 905: Lysyl oxidase is required for chemotaxis

Abstract Purpose: Lysyl oxidase (LOX) increases extracellular matrix stiffness by cross-linking collagen. A stiffer matrix drives integrin activation, and therefore LOX is believed to promote cancer cell invasion through enhanced integrin signaling. However increased matrix stiffness does not necessarily lead to a more permissive environment for invasion. Fibrillar collagen matrix orientation, density and porosity also determine how well cells can invade. Assays based on collagen gel-covered transwell systems have previously been used to show that LOX is required for invasion. These systems are driven by a chemoattractant gradient, and thus a role for LOX in chemotaxis could not be excluded. In this study we aimed to clarify whether LOX was required for matrix invasion or chemotaxis. Experimental procedures: To set up a 3D invasion assay that was not driven by a chemoattractant gradient, spheroids from multiple LOX expressing cancer cell lines were embedded into a thick 3D collagen gel. Cells were then allowed to randomly invade into the surrounding collagen. Alternatively, cancer cells were allowed to invade a fibroblast-modified 3D collagen gel in an organotypic assay. In this assay the invasion was driven by a strong chemoattractant gradient through a dense collagen gel, with cancer cells cultured atop the collagen gel at the air-medium interface. A Dunn chemotaxis chamber or a transwell tissue culture insert was used to investigate chemotaxis towards serum or EGF. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) specific to human LOX was used to deplete LOX expression in the cancer cells, to test potential LOX functions in the above assays. Results: LOX depletion in parental MDA-MB-231 cells, MDA-MB-231 brain or bone metastatic sub-population cells, U87 and U118 cells did not impact 3D collagen gel invasion. LOX inhibition by BAPN also did not impact collagen gel invasion in MDA-MB-231, LN229, U87, U118 and U138 cells. The data indicated that LOX was not required for invasion through a thick 3D collagen gel in vitro. However, in the chemotactic-driven organotypic assay, loss of LOX in MDA-MB-231 cells completely abolished invasion. When the chemotactic potential towards serum and EGF of LOX depleted MDA-MB-231 and U87 cells was tested using Dunn chemotaxis chambers, we observed strong chemotactic defects in both cell lines. The chemotactic defects of these cells were also observed when a transwell based chemotaxis assay was used. Random cell migration of MDA-MB-231 and U87 cells were otherwise not affected by LOX depletion. Conclusions: LOX is not required for collagen matrix invasion per se. Instead LOX is important for cells to sense chemoattractants and to maintain directional cell migration. The molecular mechanisms underlying these observations are now the subject of further investigation. Citation Format: Haoran Tang, Nathalie Dhomen, Richard Marais. Lysyl oxidase is required for chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2017-905

Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism.

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5' untranslated region (5' UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5' UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5' UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5' UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding.IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5' untranslated region (5' UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5' UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis trp operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.

Viral Delivery of dsRNA for Control of Insect Agricultural Pests and Vectors of Human Disease: Prospects and Challenges.

RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediate molecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed.

Structure and Dynamics of the Tetra-A Loop and (A-A)-U Sequence Motif within the Coliphage GA Replicase RNA Operator.

The three-dimensional structure of a RNA hairpin containing the RNA operator binding site for bacteriophage GA coat protein is presented. The phage GA operator contains the asymmetric (A-A)-U sequence motif and is capped by a four-adenine (tetra-A) loop. The uridine of the (A-A)-U motif preferentially pairs with the 5'-proximal cross-strand adenine, and the 3'-proximal adenine stacks into the helix. The tetra-A loop is well-ordered with adenine residues 2-4 forming a 3' stack. This loop conformation stands in contrast to the structure of the 5'-AUUA loop of the related phage MS2 operator in which residues 1 and 2 form a 5' stack. The context dependence of the (A-A)-U sequence motif conformation was examined using structures of 76 unique occurrences from the Protein Data Bank. The motif almost always has one adenine bulged and the other adenine adopting an A-U base pair. In the case in which the (A-A)-U motif is flanked by only one Watson-Crick base pair, the adenine adjacent to the flanking base pair tends to bulge; 80% of motifs with a 3' flanking pair have a 3' bulged adenine, and 84% of motifs with a 5' flanking pair have a 5' bulged adenine. The frequencies of 3'- and 5'-proximal adenines bulging are 33 and 67%, respectively, when the (A-A)-U motif is flanked by base pairs on both sides. Although a 3' flanking cytidine correlates (88%) with bulging of the 5'-proximal adenine, no strict dependence on flanking nucleotide identity was identified for the 5' side.