Structure and dynamics of the HIV-1 frameshift element RNA

The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs

HIV-1 replication and the cellular eukaryotic translation apparatus

Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication

Investigation Of A 16s Rna Central Domain Pseudoknot

X-ray crystallography of the prokaryotic 30S ribosomal subunit revealed a myriad of complex RNA-RNA, RNA-protein, and protein-protein interactions. Among these are several phylogenetically conserved RNA pseudoknots. Pseudoknots are structurally and functionally diverse RNA secondary structures. They are generally formed by two short complimentary sequences separated by many bases of single stranded regions or loops. These relatively simple folds are often yield complex structures that are key components of functionally important conformational changes in RNA structure. One such pseudoknot is located in the central domain of the 16S rRNA. The central domain pseudoknot is formed by Watson-Crick base pairing between G570-C866 and U571-A865. Previous studies by other groups show that formation of this pseudoknot is critical for ribosome function. To examine the role this pseudoknot in ribosome function, we constructed and assayed all 255 possible mutations at these four residues. Our data show that disruption of base pairing between positions 570-866 reduces ribosome function by approximately 50% and that mutations that disrupt pairing between 571-865 completely block protein synthesis. Ribosomal proteins S8, S11 and S5 have binding sites near the central pseudoknot. To determine if mutations in the pseudoknot affect the binding of these proteins, the genes for S8, S11 and S5 were cloned and coexpressed with representative mutations at each of the sites in the pseudoknot. No complementation was observed in any of the mutants tested, indicating that loss of function in the mutants is not due to reduction in binding of ribosomal proteins. To determine the influence of thermodynamics on the activity of the mutants, each mutant was assayed at 25°C, 30°C, 37°C, and 40°C. Mutants with no measurable protein synthesis activity at 37°C were unaffected by changes in incubation temperatures. Mutants with partial activity, however, were slightly more active at 40°C but were strongly inhibited by incubation temperatures below 37°C. These data suggest that the central pseudoknot is dynamic and may facilitate the switch between two active conformations of rRNA. Mutations in the pseudoknot may therefore create thermodynamic minima that favor one conformation over the other. Homology modeling and ribosome profiles suggest that the mutations may affect ribosome association or one of the partial reactions during the protein synthesis process itself

Stability and Kinetics of DNA Pseudoknots: Formation of T∗A•T Base-Triplets and Their Targeting Reactions

Pseudoknots have been found to play important roles in the biology of RNA. These stem-loop motifs are considered to be very compact and the targeting of their loops with complementary strands is accompanied with lower favorable free energy terms. We used a combination of spectroscopic (UV, CD and fluorescence), calorimetric (DSC, PPC and ITC) and kinetic (SPR) techniques to investigate: 1) Local base-triplet formation in pseudoknots; 2) energetic contributions for the association of pseudoknots with their complementary strands; and 3) the kinetic rates as a function of targeting strand length. We investigated a set of DNA pseudoknots with sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where “Tn” is a thymine loop with n = 5, 7, 9, and 11. The favorable folding of each pseudoknot resulted in favorable enthalpy-entropy compensation, correlated to favorable base-pair stacking contributions and unfavorable uptakes of ions and water molecules. The increase in the length of the loop yielded higher TMs, 53°C to 59°C and folding enthalpies ranging from -60 to -110 kcal/mol, resulting in a significant stabilization, ΔG°(5) = -8.5 to -16.6 kcal/mol, which is consistent with the formation of 1-2 TAT/TAT base-triplet stacks. The PPC results yielded folding volume changes, ΔVs, ranging from 18 to 23 ml/mol, indicating the higher volume of the folded pseudoknots is due to the uptake of both water (ΔnW of -11 to -24 mol H2O/mol) and ions (Δnion of -2.5 to -4.1 mol Na+/mol). We use ITC and DSC to determine thermodynamic profiles for the reaction of pseudoknots with partially complementary strands. We obtained favorable reaction free energies terms. However, the targeting of compact pseudoknots containing local base-triplets is less favorable due to their larger folding free energy term. The SPR data indicated that the rate of association, kon, decreases while the rate of dissociation, koff, increases as the length of the targeting strand increases, which yielded increasing KD, app.. This indicates the affinity of the target strand to the pseudoknot decreases as the length of the target strand increases. A similar trend was obtained when dissociation constants, KD, DSC, were measured from DSC Hess cycles. However, the KD, DSC were much smaller. This apparent discrepancy between these techniques is that SPR is measuring both the initial association and initial dissociation rates of steady state equilibrium states, while DSC measures true equilibrium states of the entire molecules

Conserved RNA Pseudoknots

Pseudoknots are essential for the functioning of many small RNA molecules. In addition, viral RNAs often exhibit pseudoknots that are required at various stages of the viral life-cycle. Techniques for detecting evolutionarily conserved, and hence most likely functional RNA pseudoknots, are therefore of interest. Here we present an extension of the alidot approach that extracts conserved secondary structures from a multiple sequence alignment and predicted secondary structures of the individual sequences. In contrast to purely phylogenetic methods, this approach yields good results already for small samples of 10 sequences or even less

Conserved RNA Pseudoknots

Pseudoknots are essential for the functioning of many small RNA molecules. In addition, viral RNAs often exhibit pseudoknots that are required at various stages of the viral life-cycle. Techniques for detecting evolutionarily conserved, and hence most likely functional RNA pseudoknots, are therefore of interest. Here we present an extension of the alidot approach that extracts conserved secondary structures from a multiple sequence alignment and predicted secondary structures of the individual sequences. In contrast to purely phylogenetic methods, this approach yields good results already for small samples of 10 sequences or even less

PF: Conserved RNA Pseudoknots

Abstract: Pseudoknots are essential for the functioning of many small RNA molecules. In addition, viral RNAs often exhibit pseudoknots that are required at various stages of the viral life-cycle. Techniques for detecting evolutionarily conserved, and hence most likely functional RNA pseudoknots, are therefore of interest. Here we present an extension of thealidot approach that extracts conserved secondary structures from a multiple sequence alignment and predicted secondary structures of the individual sequences. In contrast to purely phylogenetic methods, this approach yields good results already for small samples of 10 sequences or even less.