The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges.

BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.

Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15.

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.

The crystal structure of UUCG tetraloop.

All large structured RNAs contain hairpin motifs made of a stem closed by several looped nucleotides. The most frequent loop motif is the UUCG one. This motif belongs to the tetraloop family and has the peculiarity of being highly thermodynamically stable. Here, we report the first crystal structure of two UUCG tetraloops embedded in a larger RNA-protein complex solved at 2.8 A resolution. The two loops present in the asymmetric unit are in a different crystal packing environment but, nevertheless, have an identical conformation. The observed structure is globally close to that obtained in solution by nuclear magnetic resonance. However, subtle differences point to a more detailed picture of the role played by 2'-hydroxyl groups in stabilising this tetraloop.

Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallography.

Radiation damage in macromolecular crystals is not suppressed even at 90 K. This is particularly true for covalent bonds involving an anomalous scatterer (such as bromine) at the 'peak wavelength'. It is shown that a series of absorption spectra recorded on a brominated RNA faithfully monitor the extent of cleavage. The continuous spectral changes during irradiation preserve an 'isosbestic point', each spectrum being a linear combination of 'zero' and 'infinite' dose spectra. This easily yields a good estimate of the partial occupancy of bromine at any intermediate dose. The considerable effect on the near-edge features in the spectra of the crystal orientation versus the beam polarization has also been examined and found to be in good agreement with a previous study. Any significant influence of the (C-Br bond/beam polarization) angle on the cleavage kinetics of bromine was also searched for, but was not detected. These results will be useful for standard SAD/MAD experiments and for the emerging 'radiation-damage-induced phasing' method exploiting both the anomalous signal of an anomalous scatterer and the 'isomorphous' signal resulting from its cleavage.

Detection and treatment of twinning: an improvement and new results.

This work deals with two aspects of the twinning problem. Firstly, an improvement of a known statistical test aimed at detecting twinning is presented and, secondly, a new parametrization of twinning is described, as well as a new method to obtain an accurate estimate of the degree of twinning. During work on crystals of the dimerization-initiation site of the HIV-1 genomic RNA, perfectly twinned crystals were obtained which were not immediately recognized as such by use of a known statistical method. This method, reminiscent of Wilson tests for the detection of centrosymmetric space groups, relies on calculation of /2 or, equivalently, of /2. It is shown that overlooking experimental errors may lead to erroneously large values of this index and, in turn, to ambiguous or incorrect conclusions. An immediate solution to this problem is presented. Independently, an alternative parametrization which expresses both the effect of twinning on intensities and the operation of untwinning to recover the correct intensities is proposed. A new method for estimating the degree of twinning is also presented. It is based upon maximization of the cross-correlation coefficients between intensities of all available data sets, and yields a fully analytical solution. Tests made with experimental data are quite satisfactory. It is suggested that the latter results could be used efficiently within the MIR method by allowing refinement, through one additional parameter only, of the twinning ratios of all data sets considered for phasing. Finally, the new parametrization of twinning has striking consequences in this correlation-based twinning determination: very unexpectedly, it yields a novel estimate of the 'twinning ratio' of a potentially twinned crystal which is fully independent of the data set used for the comparison.

Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms.

The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness.

Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site.

We describe the crystal structures of the RNA dimerization initiation sites (DIS) of HIV-1 subtypes A and B. Both molecules adopt a hairpin conformation, with loop sequences consisting of 272-AGGUGCACA-280 and 272-AAGCGCGCA-280, respectively. This includes a six-base self-complementary stretch (underlined) that allows homodimerization through the formation of a loop-loop, or 'kissing-loop', complex. The DISs for the two sequences have identical conformations, and the two interacting hairpins show a perfect coaxial alignment. The conserved purines, A272 and R273, are stacked in a bulged-out conformation and symmetrically join the upward and downward strands of each hairpin by crossing the helix major groove. There is good agreement between these structures and previous results from chemical probing in solution, as well as with differences in magnesium dependence for dimerization. The overall shape of the kissing-loop complex is very similar to that of the previously determined subtype A DIS duplex form. Unexpectedly, the purine R273 is the only base seen at a different position and is responsible for the difference in topology between the two forms. We propose that the transition from kissing-loop duplex could occur by a recombination mechanism based on symmetrical chain cleavage at R273 of each hairpin and subsequent cross-religation, rather than by base-pair melting and subsequent reannealing.

X-ray-induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing.

Multi-wavelength anomalous dispersion (MAD) using brominated derivatives is considered a common and convenient technique for solving chemically synthesized nucleic acid structures. Here, it is shown that a relatively moderate X-ray dose (of the order of 5 x 10(15) photons mm(-2)) can induce sufficient debromination to prevent structure determination. The decrease in bromine occupancy with radiation dose can be accounted for by a simple exponential, with an estimated rate constant at the absorption-peak wavelength, 7.4 (0.8) MGy, that is not significantly different from its value at the absorption-edge wavelength, 9.2 (2.6) MGy (the given e.s.d.s assess the relative closeness of the two values, not their absolute accuracy, which is probably worse). Chemically, these results (and others) are consistent with bromine cleavage resulting from direct photodissociation and/or from the action of free electrons, rather than from the action of hydroxyl radicals originating from water dissociation. The free bromine species (Br(-)) diffuse too quickly, even in amorphous ice around 100 K, to allow the determination of a diffusion coefficient. From a practical point of view, it is suggested that a single data collection with a crystal consisting of iodinated instead of brominated derivatives could provide both anomalous scattering and SIR phase information by the progressive cleavage of iodine.

Crystal structure of the S15-rRNA complex.

In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 ¿ resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.