The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability

The crystal and molecular structure of an RNA duplex corresponding to the high affinity Rev protein binding element (RBE) has been determined at 2.1-Å resolution. Four unique duplexes are present in the crystal, comprising two structural variants. In each duplex, the RNA double helix consists of an annealed 12-mer and 14-mer that form an asymmetric internal loop consisting of G-G and G-A noncanonical base pairs and a flipped-out uridine. The 12-mer strand has an A-form conformation, whereas the 14-mer strand is distorted to accommodate the bulges and noncanonical base pairing. In contrast to the NMR model of the unbound RBE, an asymmetric G-G pair with N2-N7 and N1-O6 hydrogen bonding, is formed in each helix. The G-A base pairing agrees with the NMR structure in one structural variant, but forms a novel water-mediated pair in the other. A backbone flip and reorientation of the G-G base pair is required to assume the RBE conformation present in the NMR model of the complex between the RBE and the Rev peptide.

Point mutations and sequence variability in proteins: Redistributions of preexisting populations

Here we study the effect of point mutations in proteins on the redistributions of the conformational substates. We show that regardless of the location of a mutation in the protein structure and of its type, the observed movements of the backbone recur largely at the same positions in the structures. Despite the different interactions that are disrupted and formed by the residue substitution, not only are the conformations very similar, but the regions that move are also the same, regardless of their sequential or spatial distance from the mutation. This observation leads us to conclude that, apart from some extreme cases, the details of the interactions are not critically important in determining the protein conformation or in specifying which parts of the protein would be more prone to take on different local conformations in response to changes in the sequence. This finding further illustrates why proteins manifest a robustness toward many mutational events. This nonuniform distribution of the conformer population is consistently observed in a variety of protein structural types. Topology is critically important in determining folding pathways, kinetics, building block cutting, and anatomy trees. Here we show that topology is also very important in determining which regions of the protein structure will respond to sequence changes, regardless of the sequential or spatial location of the mutation.

Direct observation of the oceanic CO2 increase revisited

We show, from recent data obtained at specimen North Pacific stations, that the fossil fuel CO2 signal is strongly present in the upper 400 m, and that we may consider areal extrapolations from geochemical surveys to determine the magnitude of ocean fossil fuel CO2 uptake. The debate surrounding this topic is illustrated by contrasting reports which suggest, based upon atmospheric observations and models, that the oceanic CO2 sink is small at these latitudes; or that the oceanic CO2 sink, based upon oceanic data and models, is large. The difference between these two estimates is at least a factor of two. There are contradictions arising from estimates based on surface partial pressures of CO2 alone, where the signal sought is small compared with regional and seasonal variability; and estimates of the accumulated subsurface burden, which correlates well other oceanic tracers. Ocean surface waters today contain about 45 μmol⋅kg−1 excess CO2 compared with those of the preindustrial era, and the signal is rising rapidly. What limits should we place on such calculations? The answer lies in the scientific questions to be asked. Recovery of the fossil fuel CO2 contamination signal from analysis of ocean water masses is robust enough to permit reasonable budget estimates. However, because we do not have sufficient data from the preindustrial ocean, the estimation of the required Redfield oxidation ratio in the upper several hundred meters is already blurred by the very fossil fuel CO2 signal we seek to resolve.

Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins.

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with G1, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA (Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that for wild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.

Geochemical challenge to earthquake prediction.

The current status of geochemical and groundwater observations for earthquake prediction in Japan is described. The development of the observations is discussed in relation to the progress of the earthquake prediction program in Japan. Three major findings obtained from our recent studies are outlined. (i) Long-term radon observation data over 18 years at the SKE (Suikoen) well indicate that the anomalous radon change before the 1978 Izu-Oshima-kinkai earthquake can with high probability be attributed to precursory changes. (ii) It is proposed that certain sensitive wells exist which have the potential to detect precursory changes. (iii) The appearance and nonappearance of coseismic radon drops at the KSM (Kashima) well reflect changes in the regional stress state of an observation area. In addition, some preliminary results of chemical changes of groundwater prior to the 1995 Kobe (Hyogo-ken nanbu) earthquake are presented.