Quasi-continuous Helix Research Articles

Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of −1 ribosomal frameshifting

-1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient -1 PRF, two cis-acting elements are required: a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. We have analyzed the gag-pol junction sequences from 4254 HIV-1 strains. Approximately ninety-five percent of the sequences can form four pseudoknots PK1-PK4 (∼ 97% contain PK1, PK3, and PK4), covering ∼ 72 nt including the frameshift site. Some pseudoknots are mutually excluded due to sequence overlap. PK1 and PK3 arrange tandemly. Their stems form a quasi-continuous helix of ∼ 22 bp. We propose a novel mechanism for possible roles of these pseudoknots. Multiple alternative structures may exist at the gag-pol junction. In most strains, the PK1-PK3 tandem pseudoknots may dominate the structurally heterogeneous pool of RNA due to their greater overall stability. The tandem pseudoknots may function as a breaking system to slow down the ribosome. The ribosome unwinds PK1 and stem 1 of PK3 before it can reach the frameshift site. Then, PK4 can form rapidly because the intact stem 2 of PK3 makes up a large part of the stem 1 of PK4. The newly formed PK4 jams the entrance of the mRNA tunnel. The process then proceeds as in a typical case of -1 PRF. This mechanism incorporates several exquisite new features while still being consistent with the current paradigm of pseudoknot-dependent -1 PRF.

NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer.

This work presents the first structural analysis of an RNA-DNA complex consisting of an 18 nt RNA hairpin and a 20 nt DNA aptamer. The DNA molecule was previously selected, from a randomly synthesized library, against the transactivation response element (TAR) involved in transcriptional regulation of the HIV genome. The DNA aptamer used in the present study is an imperfect stem-loop with the sequence 5'-ACTCCCAT-3', characteristic of the selected candidates, in the apical loop. This octameric motif contains five bases complementary to the TAR loop sequence 5'-CUGGGA-3'. The use of homo- and heteronuclear NMR spectroscopy allowed assignment of the complex resonances and resolution of its secondary structure. Evidence is given for a kissing complex fold, which consists of a quasi-continuous helix formed by one stem of DNA, one stem of RNA and a central hybrid helix comprising 5 bp. Two out of helices residues of DNA and one of RNA connect the DNA-RNA loop-loop helix to the stem of either partner in the complex. In addition, two thymines of the DNA stem are engaged in a non-canonical T.T base pair.

A conformational switch at the 3' end of a plant virus RNA regulates viral replication.

3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 Å crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson–Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA.

. The translation elongation factor EF-Tu in its GTP-bound state forms a ternary complex with any aminoacylated tRNA (aa-tRNA), except initiator tRNA and selenocysteinyl-tRNA. This complex delivers aa-tRNA to the ribosomal A site during the elongation cycle of translation. The crystal structure of the yeast Phe-tRNAPhe ternary complex with Thermus aquaticus EF-Tu-GDPNP (Phe-TC) has previously been determined as one representative of this general yet highly discriminating complex formation. The ternary complex of Escherichia coli Cys-tRNACys and T. aquaticus EF-Tu-GDPNP (Cys-TC) has been solved and refined at 2.6 degrees resolution. Conserved and variable features of the aa-tRNA recognition and binding by EF-Tu-GTP have been revealed by comparison with the Phe-TC structure. New tertiary interactions are observed in the tRNACys structure. A 'kissing complex' is observed in the very close crystal packing arrangement. The recognition of Cys-tRNACys by EF-Tu-GDPNP is restricted to the aa-tRNA motif previously identified in Phe-TC and consists of the aminoacylated 3' end, the phosphorylated 5' end and one side of the acceptor stem and T stem. The aminoacyl bond is recognized somewhat differently, yet by the same primary motif in EF-Tu, which suggests that EF-Tu adapts to subtle variations in this moiety among all aa-tRNAs. New tertiary interactions revealed by the Cys-tRNACys structure, such as a protonated C16:C59 pyrimidine pair, a G15:G48 'Levitt pair' and an s4U8:A14:A46 base triple add to the generic understanding of tRNA structure from sequence. The structure of the 'kissing complex' shows a quasicontinuous helix with a distinct shape determined by the number of base pairs.

RNA-RNA interactions between oligonucleotide substrates for aminoacylation

RNA stem-loop microhelices with helix sequences based on tRNA acceptor stems can be charged with specific amino acids. Experiments were designed to test the possibility that microhelices could laterally associate through complementary loop sequences and thereby bring their attached aminoacyl groups close enough together to form a peptide bond. Computer simulations suggested that formation of such complexes would be sensitive to the number of loop nucleotides needed to span the grooves of the quasi-continuous helix of the intermolecular pseudoknot so formed. These predictions were confirmed experimentally by observation of complex formation sensitivity to loop size. Complexes with optimized loop sizes had apparent bimolecular dissociation constants of approximately 100 nM with only three complementary base pairs between the respective loops. Single nucleotide substitutions that disrupted the predicted intermolecular loop-loop base-pairing abolished detectable association. Similarly, placing a gap between the short helix formed by loop-loop pairing and the adjacent acceptor stems also diminished complex formation. These experiments establish an experimental basis for microhelix association for peptide synthesis.

The structure of an RNA “kissing” hairpin complex of the HIV TAR hairpin loop and its complement

We have used nuclear magnetic resonance (NMR) to obtain the structure of an RNA “kissing” hairpin complex formed between the HIV-2 TAR hairpin loop and a hairpin with a complementary loop sequence. Kissing hairpins are important in natural antisense reactions; their complex is a specific target for protein binding. The complex has all six nucleotides of each loop paired to form a bent quasicontinuous helix of three coaxially stacked helices: two stems plus a loop-loop interaction helix. Experimental constraints derived from heteronuclear and homonuclear NMR data on 13C and 15N-labeled RNA led to a structure for the loop-loop helix with an average root-mean-square deviation of 0.83 (±0.10) Å for 33 converged structures relative to the average structure. The loop-loop helix of the kissing complex is distorted compared to A-form RNA. Its major groove is blocked by the phosphodiester bonds that connect the first loop residue of each hairpin with its own stem, and it is flanked by two negatively charged phosphate clusters. The loop-loop helix has alternating helical twists between adjacent base-pairs. The base-pairs at the helix junctions are overwound and three base-pairs near the helix junctions adopt high propeller twists. All these changes reduce the distance needed for the bridging phosphodiester bonds connecting each stem and loop to cross the major groove of the loop-loop helix, and result in a deformed RNA helix with localized perturbations in the minor groove surface. The alternating helical twist pattern, plus other distortions in the loop-loop helix may be important for Rom protein recognition of the kissing hairpin complex.

Structural features of a three-stranded DNA junction containing a C-C junctional bulge.

We have examined the stability of junctional base pairs in a three-way DNA junction with two unpaired cytidine residues at the branch point using two-dimensional nuclear Overhauser effect spectroscopy in H2O solution. Our data directly support the presence of two of the three junctional Watson-Crick base pairs, with indirect support for the third as well. These results complement the data presented in the preceding paper, where we examined the nonexchangeable proton resonance assignments of three-way DNA junctions from NOESY data in D2O solution. We have incorporated the NOE data from both sets of experiments, using this information as input for a combined distance geometry (DG) and simulated annealing (SA) protocol designed to derive three-dimensional structures of the junction molecule consistent with the NMR data. Although the data does not allow us to derive a unique solution for the structure of the molecule, certain conformational features are invariably present in our models. We demonstrate the existence of a preferred, pair-wise stacking arrangement between two of the three helices in the junction. Furthermore, the remaining duplex stem is situated so that it always forms an acute angle with just one of the arms from the quasi-continuous helix. The unpaired residues provide an extended backbone segment linking two of the helices together. The first unpaired base on the 5' end loops out from the interior of the molecule to reside along the minor groove of one helix. The second is located within the interior of the molecule, stacking below one of the junctional base pairs. Our findings suggest that junctional base pair stacking is an important determinant in the conformation of multistranded nucleic acid junctions. In three-way junctions, the presence of unpaired bases at the branch point provides a relief from covalent constraints that would otherwise prevent the simultaneous realization of both base pairing and base pair stacking within the branch point of the molecule.

Secondary structure at the 3′ terminal region of RNA coliphages: Comparison with tRNA

Secondary structure models for the 3′ non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes. We report the following observations. (1) In contrast to the coding regions, the structure at the 3′ terminus is characterized by stable regular helices. We note the occurrence of the loop sequences 5′-GUUCGC and 5′-CGAAAG, that are reported to confer exceptional stability to stem structures. These features are probably present to promote the segregation of mother and daughter strands during replication. (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability. (3) We have compared the structure of phage RNA with tRNA. Overall similarity is low, but one common element may exist. It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the TψC arm and the aminoacyl acceptor arm in tRNA. As in tRNA, this structure element starts after the fourth nucleotide from the 3′ end. (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix. We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA. The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein. This common binding site may serve as a switch from replication to packaging.

Nonintercalative binding of proflavin to Z-DNA: structure of a complex between d(5BrC-G-5BrC-G) and proflavin.

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.

Structure of a Z-DNA with two different backbone chain conformations. Stabilization of the decadeoxyoligonucleotide d(CGTACGTACG) by [Co(NH3)6]3+ binding to the guanine.

The complex between cobalt hexammine and decadeoxyoligomer d(CGTACGTACG) crystallizes into the space group P65 with unit cell constants a = b = 17.93A, and c = 43.41A. The molecules have the helix axis coincident with the crystal c-axis. The decamers stack on top of each other and form a quasi-continuous helix. The structure is disordered. The asymmetric unit is a dimer (pPyr-pPur)2 with each base pair 60% of the time a C-G and 40% of the time a T-A. Restrainted least-squares refinement led to an R-factor of 25.5% for 506 observed reflections above the two-sigma level. The structure was found to have one strand in the ZI-conformation and the other in the ZII-conformation. The cobalt hexammine binds to two ZII-chains of symmetrically related molecules. On one ZII chain, two ammonia molecules of the cobalt hexammine bind to the N7 nitrogen and 06 oxygen atoms of the guanine bases and a third ammonia to the phosphate anionic oxygen atom of the preceding pyrimidine base, resulting in an "external" binding mode. On the other ZII chain, one ammonia molecule of the cobalt hexammine binds only to the anionic oxygens of the phosphate group of the guanine bases, leading to an "internal" binding mode. Thus, the basis of the stabilization of Z-DNA by [Co(NH3)6]3+ is its binding to only guanine nucleotides. It is surmised that statistical disordering of deoxyoligonucleotide structures which take a Z conformation, depends on the length of the oligomer. That is to say, octamers and decamers (which cannot use an integral number of molecules for a 12 base pair repeat) form disordered structures whereas tetramers and hexamers form well ordered structures.