Mutational analysis of the RNA pseudoknot involved in efficient ribosomal frameshifting in simian retrovirus-1.

Mutational effects on frameshifting efficiency of the RNA pseudoknot involved in ribosomal frameshifting in simian retrovirus-1 (SRV-1) have been investigated. The primary sequence and the proposed secondary structure of the SRV-1 pseudoknot are similar to those of other efficient frameshifting pseudoknots in mouse mammary tumor virus (MMTV) and feline immunodeficiency virus (FIV), where an unpaired adenine nucleotide intercalates between stem 1 and stem 2. In SRV-1 pseudoknot, the adenine nucleotide in between stem 1 and stem 2 has a potential to form an A*U base pair with the last uridine nucleotide in the loop 2, resulting in a continuous A-form helix with coaxially stacked stem 1 and stem 2. To test whether this A*U base pairing and coaxial stacking of stem 1 and stem 2 is absolutely required for efficient frameshifting in SRV-1, a series of mutants changing this potential A.U base pair to either G.C base pair or A.A, A.G, A.C, G.A, G.G mismatch is generated, and their frameshifting efficiencies are investigated in vitro using rabbit reticulocyte lysate translation assay. The frameshifting abilities of these mutant pseudoknots are similar to that of the wild-type pseudoknot, suggesting that the A*U base pair in between stem 1 and stem 2 is not necessary to promote efficient frameshifting in SRV-1. These results reveal that coaxial stacking of stem 1 and stem 2 with a Watson-Crick A.U base pair in between two stems is not a required structural feature of the pseudoknot for promoting efficient frameshifting in SRV-1. Our mutational data suggest that SRV-1 pseudoknot adopts similar structural features common to other efficient frameshifting pseudoknots as observed in MMTV and FIV.     

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.     

A Characteristic Bent Conformation of RNA Pseudoknots Promotes –1 Frameshifting during Translation of Retroviral RNA

The structures of four different RNA pseudoknots that provide one of the signals required for ribosomal frameshifting in mouse mammary tumor virus have been determined by NMR. The RNA pseudoknots have similar sequences and assume similar secondary structures, but show significantly different frameshifting efficiencies. The three-dimensional structures of one frameshifting and one non-frameshifting RNA pseudoknot had been determined previously by our group. Here we determine the structures of two new RNA pseudoknots, and relate the structures of all four pseudoknots to their frameshifting abilities. The two efficient frameshifting pseudoknots adopt characteristic bent conformations with stem 1 bending towards the major groove of stem 2. In contrast, the two poor frameshifting pseudoknots have structures very different from each other and from the efficient frameshifters. One has linear, coaxially stacked stems, the other has stems twisted and bent, but in the opposite direction to the efficient frameshifters. Changes in loop size that favor bending (shorter loops) increase frameshifting efficiency; longer loops that allow linear arrangement of the stems decrease frameshifting. Frameshifting pseudoknots in feline immunodeficiency virus and simian retrovirus have different loop sequences, but the sequences at their stem junctions imply the same bent conformation as in the mouse mammary tumor viral RNA. The requirement for a precise pseudoknot conformation for efficient frameshifting strongly implies that a specific interaction occurs between the viral RNA pseudoknot and the host protein-synthesizing machinery.     

The role of RNA pseudoknot stem 1 length in the promotion of efficient -1 ribosomal frameshifting.

The ribosomal frameshifting signal present in the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains a classic hairpin-type RNA pseudoknot that is believed to possess coaxially stacked stems of 11 bp (stem 1) and 6 bp (stem 2). We investigated the influence of stem 1 length on the frameshift process by measuring the frameshift efficiency in vitro of a series of IBV-based pseudoknots whose stem 1 length was varied from 4 to 13 bp in single base-pair increments. Efficient frameshifting depended upon the presence of a minimum of 11 bp; pseudoknots with a shorter stem 1 were either non-functional or had reduced frameshift efficiency, despite the fact that a number of them had a stem 1 with a predicted stability equal to or greater than that of the wild-type IBV pseudoknot. An upper limit for stem 1 length was not determined, but pseudoknots containing a 12 or 13 bp stem 1 were fully functional. Structure probing analysis was carried out on RNAs containing either a ten or 11 bp stem 1; these experiments confirmed that both RNAs formed pseudoknots and appeared to be indistinguishable in conformation. Thus the difference in frameshifting efficiency seen with the two structures was not simply due to an inability of the 10 bp stem 1 construct to fold into a pseudoknot. In an attempt to identify other parameters which could account for the poor functionality of the shorter stem 1-containing pseudoknots, we investigated, in the context of the 10 bp stem 1 construct, the influence on frameshifting of altering the slippery sequence-pseudoknot spacing distance, loop 2 length, and the number of G residues at the bottom of the 5'-arm of stem 1. For each parameter, it was possible to find a condition where a modest stimulation of frameshifting was observable (about twofold, from seven to a maximal 17 %), but we were unable to find a situation where frameshifting approached the levels seen with 11 bp stem 1 constructs (48-57 %). Furthermore, in the next smaller construct (9 bp stem 1), changing the bottom four base-pairs to G.C (the optimal base composition) only stimulated frameshifting from 3 to 6 %, an efficiency about tenfold lower than seen with the 11 bp construct. Thus stem 1 length is a major factor in determining the functionality of this class of pseudoknot and this has implications for models of the frameshift process.     

Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: a mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site

Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a -1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a -1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.     

A mutant RNA pseudoknot that promotes ribosomal frameshifting in mouse mammary tumor virus.

A single A-->G mutation that changes a potential A.U base pair to a G.U pair at the junction of the stems and loops of a non-frameshifting pseudoknot dramatically increases its frameshifting efficiency in mouse mammary tumor virus. The structure of the non-frameshifting pseudoknot APK has been found to be very different from that of pseudoknots that cause efficient frameshifting [Kang,H., Hines,J.V. and Tinoco,I. (1995) J. Mol. Biol. , 259, 135-147]. The 3-dimensional structure of the mutant pseudoknot was determined by restrained molecular dynamics based on NMR-derived interproton distance and torsion angle constraints. One striking feature of the mutant pseudoknot compared with the parent pseudoknot is that a G.U base pair forms at the top of stem 2, thus leaving only 1 nt at the junction of the two stems. The conformation is very different from that of the previously determined non-frameshifting parent pseudoknot, which lacks the A.U base pair at the top of the stem and has 2 nt between the stems. However, the conformation is quite similar to that of efficient frameshifting pseudoknots whose structures were previously determined by NMR. A single adenylate residue intervenes between the two stems and interrupts their coaxial stacking. This unpaired nucleotide produces a bent structure. The structural similarity among the efficient frameshifting pseudoknots indicates that a specific conformation is required for ribosomal frameshifting, further implying a specific interaction of the pseudoknot with the ribosome.     

The stimulatory RNA of the Visna-Maedi retrovirus ribosomal frameshifting signal is an unusual pseudoknot with an interstem element

The stimulatory RNA of the Visna-Maedi virus (VMV) -1 ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix, reminiscent of the HIV-1 frameshift-stimulatory RNA, or as an RNA pseudoknot. The pseudoknot is unusual in that it would include a 7 nucleotide loop (termed here an interstem element [ISE]) between the two stems. In almost all frameshift-promoting pseudoknots, ISEs are absent or comprise a single adenosine residue. Using a combination of RNA structure probing, site directed mutagenesis, NMR, and phylogenetic sequence comparisons, we show here that the VMV stimulatory RNA is indeed a pseudoknot, conforming closely to the modeled structure, and that the ISE is essential for frameshifting. Pseudoknot function was predictably sensitive to changes in the length of the ISE, yet altering its sequence to alternate pyrimidine/purine bases was also detrimental to frameshifting, perhaps through modulation of local tertiary interactions. How the ISE is placed in the context of an appropriate helical junction conformation is not known, but its presence impacts on other elements of the pseudoknot, for example, the necessity for a longer than expected loop 1. This may be required to accommodate an increased flexibility of the pseudoknot brought about by the ISE. In support of this, (1)H NMR analysis at increasing temperatures revealed that stem 2 of the VMV pseudoknot is more labile than stem 1, perhaps as a consequence of its connection to stem 1 solely via flexible single-stranded loops.     

An NMR and mutational study of the pseudoknot within the gene 32 mRNA of bacteriophage T2: insights into a family of structurally related RNA pseudoknots.

NMR methods were used to investigate a series of mutants of the pseudoknot within the gene 32 messenger RNA of bacteriophage T2, for the purpose of investigating the range of sequences, stem and loop lengths that can form a similar pseudoknot structure. This information is of particular relevance since the T2 pseudoknot has been considered a representative of a large family of RNA pseudoknots related by a common structural motif, previously referred to as 'common pseudoknot motif 1' or CPK1. In the work presented here, a mutated sequence with the potential to form a pseudoknot with a 6 bp stem2 was shown to adopt a pseudoknot structure similar to that of the wild-type sequence. This result is significant in that it demonstrates that pseudoknots with 6 bp in stem2 and a single nucleotide in loop1 are indeed feasible. Mutated sequences with the potential to form pseudoknots with either 5 or 8 bp in stem2 yielded NMR spectra that could not confirm the formation of a pseudoknot structure. Replacing the adenosine nucleotide in loop1 of the wild-type pseudoknot with any one of G, C or U did not significantly alter the pseudoknot structure. Taken together, the results of this study provide support for the existence of a family of similarly structured pseudoknots with two coaxially stacked stems, either 6 or 7 bp in stem2, and a single nucleotide in loop1. This family includes many of the pseudoknots predicted to occur downstream of the frameshift or readthrough sites in a significant number of viral RNAs.     

The global structures of a wild-type and poorly functional plant luteoviral mRNA pseudoknot are essentially identical

The helical junction region of a -1 frameshift stimulating hairpin-type mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) is characterized by a novel C27.(G7-C14) loop 2-stem 1 minor groove base triple, which is stacked on a C8+.(G12-C28) loop 1-stem 2 major groove base triple. Substitution of C27 with adenosine reduces frameshifting efficiency to a level just twofold above the slip-site alone. Here, we show that the global structure of the C27A ScYLV RNA is nearly indistinguishable from the wild-type counterpart, despite the fact that the helical junction region is altered and incorporates the anticipated isostructural A27.(G7-C14) minor groove base triple. This interaction mediates a 2.3-A displacement of C8+ driven by an A27 N6-C8+ O2 hydrogen bond as part of an A(n-1).C+.G-Cn base quadruple. The helical junction regions of the C27A ScYLV and the beet western yellows virus (BWYV) pseudoknots are essentially superimposable, the latter of which contains an analogous A25.(G7-C14) minor groove base triple. These results reveal that the global ground-state structure is not strongly correlated with frameshift stimulation and point to a reduced thermodynamic stability and/or enhanced kinetic lability that derives from an altered helical junction architecture in the C27A ScYLV RNA as a significant determinant for setting frameshifting efficiencies in plant luteoviral mRNA pseudoknots.     

Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed -1 ribosomal frameshifting.

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseudoknot from the gag-pro junction of mouse intracisternal A-type particles (mIAP), an endogenous retrovirus, has been determined through analysis of dual optical wavelength, equilibrium thermal melting profiles and differential scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot proposed to have structural features in common with the gag-pro frameshifting pseudoknots from simian retrovirus-1 (SRV-1) and mouse mammary tumor virus (MMTV). In particular, the mIAP pseudoknot is proposed to contain an unpaired adenosine base at the junction of the two helical stems (A15), as well as one in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins and compensatory base-pair substitutions incorporated into helical stem 2 was used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in two closely coupled low-enthalpy transitions at low tmin which the stem 3' to A35, unfolds first, followed by unfolding of the remainder of the helical stem. The third unfolding transition is associated with some type of stacking interactions in the stem 1 hairpin loop not present in the pseudoknot. The fourth transition is assigned to unfolding of stem 1. In all RNAs investigated, DeltaHvH approximately DeltaHcal, suggesting that DeltaCpfor unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the stem 2 unfolding transition in the context of both the wild-type and DeltaA35 mutant RNAs only slightly, by DeltaDeltaG degrees approximately 1 kcal mol-1(at 37 degrees C). The DeltaA15 RNA is considerably more susceptible to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabilization of the molecule. Interestingly, substitution of the nine loop 2 nucleotides with uridine residues induces a more pronounced destabilization of the molecule (DeltaDeltaG degrees approximately 2.0 kcal mol-1), a long-range, non-nearest neighbor effect. These findings provide the thermodynamic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability.     

Crystal structure of a luteoviral RNA pseudoknot and model for a minimal ribosomal frameshifting motif

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter. Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.     

Thermodynamic analysis of conserved loop-stem interactions in P1-P2 frameshifting RNA pseudoknots from plant Luteoviridae

The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed -1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m(5)-C(8)-substituted BWYV RNA is employed to establish that the BWYV P1-P2 pseudoknot is protonated at cytidine 8 in loop L1 (delta(N(3)H)+ = 12.98 ppm), which stabilizes a C(+.)(G-C) major groove base triple by Delta(DeltaG(37))(protonation) = 3.1 (+/-0.4) kcal mol(-1). The stabilities of both the PLRV and PEMV-1 P1-P2 pseudoknots are also strongly pH-dependent, with Delta(DeltaG(37))(protonation) = 2.1 (+/-0.2) kcal mol(-1) for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by DeltaH > or = 30 kcal mol(-)(1) in excess of secondary structure predictions, attributed to loop L2-stem S1 minor groove triplex interactions. BWYV RNAs containing single 2'-deoxy or A --> G substitutions that disrupt L2-S1 hydrogen bonding are strongly destabilized with Delta(DeltaG(37))(folding) (pH = 7.0) ranging from approximately 1.8 (+/-0.3) to > or =4.0 kcal mol(-1), relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1-S2 and L2-S1 loop-stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.     

Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.     

Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 A crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson-Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na(+). Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem tertiary structural interactions which stabilize the molecule, contributing a net of DeltaH approximately -30 kcal mol(-1) and DeltaG degrees (37) of -3.3 kcal mol(-1) to a total DeltaH and DeltaG degrees (37) of -121 and -16 kcal mol(-1), respectively, at pH 6.0, 0.5 M K(+) by DSC. Characterization of mutant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-triple (pK(a)=6.8), protonation of which contributes nearly -3.5 kcal mol(-1) in net stability in the presence of a wild-type loop 2. Substitution of the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K(+), NH(4)(+) and Na(+) on other simple duplex and pseudoknotted RNAs.     

A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal

A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.     

Functional analysis of the SRV-1 RNA frameshifting pseudoknot

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting.     

Characterization of the mechanical unfolding of RNA pseudoknots

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause −1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg²⁺ significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that −1 frameshifting depends on the difficulty of unfolding the mRNA.     

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.     

Identification of novel ligands for the RNA pseudoknot that regulate -1 ribosomal frameshifting

In many viruses, -1 ribosomal frameshifting (-1RF) regulates synthesis of proteins and is crucial for virus production. An RNA pseudoknot is one of the essential components of the viral -1RF system. Thermodynamic or kinetic control of pseudoknot folding may be important in regulating the efficiency of -1RF. Thus, small molecules that interact with viral RNA pseudoknots may disrupt the -1RF system and show antiviral activity. In this study, we conducted virtual screening of a chemical database targeting the X-ray crystal structure of RNA pseudoknot complexed with biotin to identify ligands that may regulate an -1RF system containing biotin-aptamer as an RNA pseudoknot component. After docking screening of about 80,000 compounds, 58 high-ranked hits were selected and their activities were examined by in vitro and cell-based -1 frameshifting assays. Six compounds increased the efficiency of -1 frameshifting, and these are novel small molecule compounds that regulate the -1RF.     

An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus

The −1 ribosomal frameshifting requires the existence of an in cis RNA slippery sequence and is promoted by a downstream stimulator RNA. An atypical RNA pseudoknot with an extra stem formed by complementary sequences within loop 2 of an H-type pseudoknot is characterized in the severe acute respiratory syndrome coronavirus (SARS CoV) genome. This pseudoknot can serve as an efficient stimulator for −1 frameshifting in vitro. Mutational analysis of the extra stem suggests frameshift efficiency can be modulated via manipulation of the secondary structure within the loop 2 of an infectious bronchitis virus-type pseudoknot. More importantly, an upstream RNA sequence separated by a linker 5′ to the slippery site is also identified to be capable of modulating the −1 frameshift efficiency. RNA sequence containing this attenuation element can downregulate −1 frameshifting promoted by an atypical pseudoknot of SARS CoV and two other pseudoknot stimulators. Furthermore, frameshift efficiency can be reduced to half in the presence of the attenuation signal in vivo. Therefore, this in cis RNA attenuator represents a novel negative determinant of general importance for the regulation of −1 frameshift efficiency, and is thus a potential antiviral target.