Five-way Junction Research Articles

DNA nanotechnology for nucleic acid analysis: sensing of nucleic acids with DNA junction-probes.

DNA nanotechnology deals with the design of non-naturally occurring DNA nanostructures that can be used in biotechnology, medicine, and diagnostics. In this study, we introduced a nucleic acid five-way junction (5WJ) structure for direct electrochemical analysis of full-length biological RNAs. To the best of our knowledge, this is the first report on the interrogation of such long nucleic acid sequences by hybridization probes attached to a solid support. A hairpin-shaped electrode-bound oligonucleotide hybridizes with three adaptor strands, one of which is labeled with methylene blue (MB). The four strands are combined into a 5WJ structure only in the presence of specific DNA or RNA analytes. Upon interrogation of a full-size 16S rRNA in the total RNA sample, the electrode-bound MB-labeled 5WJ association produces a higher signal-to-noise ratio than electrochemical nucleic acid biosensors of alternative design. This advantage was attributed to the favorable geometry on the 5WJ nanostructure formed on the electrode's surface. The 5WJ biosensor is a cost-efficient alternative to the traditional electrochemical biosensors for the analysis of nucleic acids due to the universal nature of both the electrode-bound and MB-labeled DNA components.

Hierarchical Folding of the rRNA in the Early Assembly of the E. Coli Ribosomal Small Subunit

In order to investigate the co-transcriptional folding and assembly of the E. coli ribosomal small subunit (SSU), we applied the structure-based Go model to fold 16S rRNA fragments with increasing length. The first fragment examined is the binding domain of S20, which includes the first few local and non-local helices to be transcribed (h6, h7-h10) in vivo. The simulations show that the main barriers to the formation of the S20 binding domain are the docking of the 3' and 5' strands of the non-local h7 followed by re-orientation of the helices in the four-way junction to allow tertiary contacts between h6 and h8 to be established. These observations suggest the role of the primary binding protein S20 in stabilizing key tertiary contacts and speeding up the protein:RNA recognition during the assembly process.Transcription of rRNA then proceeds to the local five-way junction (5WJ), which nucleates the assembly of the 5' domain upon interaction with the initiator and primary binding protein S4. This complex contains the largest contiguous ribosomal structure signature, helix h16, which is a conserved feature in all bacterial 16S rRNAs. We use all-atom molecular dynamics simulations to demonstrate that the co-evolving signature in the N-terminus of S4 is intrinsically disordered and capable of accelerating the 5WJ:S4 binding process through a fly-casting mechanism. The scenario is further confirmed using the structure-based Go model for protein:RNA, with which hundreds of simultaneous folding and binding events between the 5WJ and S4 are captured. Based on analysis of both explicit solvent MD and structure-based Go trajectories, we identify multiple metastable states of the 5WJ, and various protein-guided folding pathways that compare directly with the FRET experiments.

Structural Intermediates and Folding Events in the Early Assembly of the Ribosomal Small Subunit

Using all-atom explicit solvent molecular dynamics (MD) simulations, we investigated the early structural intermediates of the 5' domain of the 16S rRNA in Escherichia coli upon the removal of the primary binding r-proteins S4, S17, and S20 and the secondary binding r-protein S16. Removal of each r-protein corresponded to the disappearance of subdomains with correlated dynamics. Correlation-based network analysis of the MD trajectories of the naked rRNA showed that the different subdomains are connected via multiple pathways with high betweenness. These pathways cross at the internal loop of helix 17 (h17) in the five-way junction (5WJ). The structure of the internal loop is disrupted by the binding of S17 and rescued by the addition of S16, suggesting an important function of the secondary binding protein in biasing the rRNA folding landscape toward the native basin. Using structure-based Gō simulations, we investigated the folding barriers of the lower four-way junction (4WJ) with h6, which is the primary binding site of S20 and the first to be transcribed. The folding of the 4WJ is consistent with the protection patterns observed in hydroxyl radical footprinting. Results from the all-atom simulations show that the fluctuations in the 5WJ are independent of the fluctuations in the 4WJ, suggesting that the subdomains fold independently and are stabilized by primary r-proteins.

How R-Proteins Stabilize the R-RNA during Early Stages of Bacterial Ribosome Biogenesis

Ribosome biogenesis in E. coli has been extensively studied, yet, very little is known about the structural changes that occur to the rRNA upon r-protein binding. Recent cryo-electron microscopy and mass spectrometry studies from the Williamson laboratory have produced a time-resolved assembly map of the 16S subunit. The first r-protein to nucleate 16S folding, S4, binds to h16-h18 of the five-way junction–a signature region that idiosyncratically separates Bacteria and Archaea. S4, subsequently, nucleates the formation of the 5' domain of the 16S and recruits other r-proteins to bind.Guided by in vitro studies, we probe how S4 nucleates the 5' domain formation using a series of all-atom molecular dynamics (MD) simulations on the E. coli 5' domain with various r-proteins systematically bound to the rRNA. Large dynamic fluctuations in the structure of the rRNA were observed in both our long-time MD trajectories and multi-color single molecule FRET experiments from our collaborators. For example, with only S4 bound to the rRNA, an A-minor interaction between h8 and h14 opens up, but is stable upon adding S17 to a location distant from h8 and h14.We also are exploring the cooperative effects of the r-protein binding by generating weighted networks based on position correlations. Graph of the network weighed by edge-betweenness, a measure of the number of shortest paths that pass through a given edge, show a continuous path connecting the binding sites of S4, S17, and S20 with a branching point located near h15–the binding site the secondary binding r-protein S16. This path is hypothesized to modulate the binding between primary and secondary binding r-proteins and further probed using additional all-atom and structure-based models.

Assembly of the Five-Way Junction in the Ribosomal Small Subunit Using Hybrid MD-Go̅ Simulations

Assembly of the bacterial ribosomal small subunit (SSU) begins with the folding of the five-way junction upon interaction with the primary binding protein S4. This complex contains the largest contiguous molecular signature, which is a conserved feature in all bacterial 16S rRNAs. In a previous study, we used all-atom molecular dynamics simulations to demonstrate that the co-evolving signature in the N-terminus of S4 is intrinsically disordered and capable of accelerating the binding process through a fly casting mechanism. In this paper, comparisons between the all-atom MD simulations and FRET experiments identify multiple metastable conformations of the naked five-way junction without the presence of S4. Furthermore, we capture the simultaneous folding and binding of the five-way junction and r-protein S4 by use of a structure-based Gō potential implemented within the framework of the all-atom molecular dynamics CHARMM force field. Different folding pathways are observed for the refolding of the five-way junction upon partial binding of S4. Our simulations illustrate the complex nature of RNA folding in the presence of a protein binding partner and provide insight into the role of population shift and the induced fit mechanisms in the protein:RNA folding and binding process.

Slow Formation of Stable Complexes during Coincubation of Minimal rRNA and Ribosomal Protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.

Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

Functional Role of Ribosomal Signatures

Although structure and sequence signatures in ribosomal RNA and proteins are defining characteristics of the three domains of life and instrumental in constructing the modern phylogeny, little is known about their functional roles in the ribosome. In this work, the largest coevolving RNA/protein signatures in the bacterial 30S ribosome are investigated both experimentally and computationally through all-atom molecular-dynamics simulations. The complex includes the N-terminal fragment of the ribosomal protein S4, which is a primary binding protein that initiates 30S small subunit assembly from the 5′ domain, and helix 16 (h16), which is part of the five-way junction in 16S rRNA. Our results show that the S4 N-terminus signature is intrinsically disordered in solution, whereas h16 is relatively stable by itself. The dynamic disordered property of the protein is exploited to couple the folding and binding process to the five-way junction, and the results provide insight into the mechanism for the early and fast binding of S4 in the assembly of the ribosomal small subunit.

A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

The lysine riboswitch is associated to the lysC gene in Bacillus subtilis, and the binding of lysine modulates the RNA structure to allow the formation of an intrinsic terminator presumably involved in transcription attenuation. The complex secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that undergoes structural changes upon ligand binding. Using single-round transcription assays, we show that a loop-loop interaction is important for lysine-induced termination of transcription. Moreover, upon close inspection of the secondary structure, we find that an unconventional kink-turn motif is present in one of the stems participating in the loop-loop interaction. We show that the K-turn adopts a pronounced kink and that it binds the K-turn-binding protein L7Ae of Archaeoglobus fulgidus in the low nanomolar range. The functional importance of this K-turn motif is revealed from single-round transcription assays, which show its importance for efficient transcription termination. This motif is essential for the loop-loop interaction, and consequently, for lysine binding. Taken together, our results depict for the first time the importance of a K-turn-dependent loop-loop interaction for the transcription regulation of a lysine riboswitch.

Evolution of High-Branching Deoxyribozymes from a Catalytic DNA with a Three-Way Junction

Here, we report the evolution of two star-shaped (five-way junction) deoxyribozymes from a catalytic DNA containing a three-way junction scaffold. The transition was shown to be a switch rather than a gradual progression. The star-shaped motifs, surprisingly, only took five selection cycles to be detected, and another four to dominate the evolving population. Chemical probing experiments indicated that the two deoxyribozymes belong to the same family despite noticeable variations in both the primary sequence and the secondary structure. Our findings not only describe the evolution of high-branching nucleic acid structures from a low-branching catalytic module, but they also illustrate the idea of deriving a rare structural motif by sampling the sequence variants of a given functional nucleic acid.

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.