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Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.

The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N-and C-terminal autoprocessing sites, by recognizing P4-P1 and P1′. In this study, we determined the crystal structure of 3CL pro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3′ position [Phe(P3′)] is snugly accommodated in the S3′ pocket. Mutations of Phe(P3′) impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C-and N-terminal sites, as follows. The S3′ subsite is formed by Phe(P2)-induced conformational changes of 3CL pro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3′ subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3′). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe (P2) exhibits a much smaller inhibitory effect, because Phe(P3′) is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).SARS | 3CL protease | specificity | subsite cooperativity | crystal structure

Quantitative polarity scales have been of great value in advancing our understanding of chemical and physical processes in bulk solvents. However, an understanding of the polarity at liquid interfaces has been more elusive and no scale of interface polarity currently exists. Following our demonstration that second harmonic spectroscopy can be used to measure interface polarity, we have determined the polarity of several liquid/ liquid and vapor/liquid interfaces. The polarities of the water/1,2-dichloroethane and water/chlorobenzene interfaces have been investigated using the polarity indicator molecule N,N-diethyl-p-nitroaniline (DEPNA). The betaine dye 4-(2,4,6-triphenylpyridinium)-2,6-diphenylphenoxide, (E T (30)), was used to probe the polarity of the air/water interface. The intramolecular charge transfer (CT) absorption band positions of both DEPNA and E T (30) are measured and used to define the interface solvent polarity. An important finding is that the polarity of the liquid interfaces is simply related to the polarity of the bulk phases. The interface polarity is found to be equal to the arithmetic average of the polarity of the adjoining bulk phases. This surprisingly simple result suggests the possible dominance of the long-range solute-solvent interactions, not the local interface interactions, in determining the difference in the excited-and ground-state solvation energies of the interface adsorbed molecules.

A spectroscopic method, based on the interface selectivity of second-harmonic generation, is used to obtain the polarity of liquid interfaces. In this paper the second-harmonic measurement of the spectrum of the polarity indicator molecule N, N′-diethyl-p-nitroaniline (DEPNA) at the air/water interface demonstrates the method. Two different approaches are used to measure the intramolecular charge transfer (CT) absorption band position of DEPNA at the air/water interface. The DEPNA CT band blue-shifts from 429 nm in bulk water (polar solvent) to 359 nm in bulk hexane (nonpolar solvent) and 329 nm in the gas phase (no solvent). At the air/water interface, the charge transfer peak band maximum occurs at 373 nm, which indicates that the polarity of the air/water interface is similar to those of the bulk solvents carbon tetrachloride and butyl ether. The DEPNA results together with the results from another solvatochromic polarity indicator molecule, ET-(30), which will be reported elsewhere, show that the polarity results of the air/water interface are general.

A new application of second harmonic generation to selectively probe the surface of microscopic size centrosymmetric structures in centrosymmetric bulk media has been applied to polymer beads in aqueous solution and to oil droplets in an oil/water emulsion. The free energies of adsorption and the surface densities of organic molecules adsorbed from aqueous solution to 1 µm polystyrene bead surfaces and to 230 nm tetradecane oil droplet surfaces have been determined. The potential use of this method as a chemical and biochemical sensor without the need for a fluorescent or magnetic tag is noted.

The ability to achieve sub-wavenumber resolution (0.6 cm(-1)) and a large signal-to-noise ratio in high-resolution broadband sum-frequency generation vibrational spectroscopy (HR-BB-SFG-VS) allows for the detailed SFG spectral lineshapes to be used in the unambiguous determination of fine spectral features. Changes in the structural spectroscopic phase in SFG-VS as a function of beam polarization and experimental geometry proved to be instrumental in the identification of an unexpected 2.78 ± 0.07 cm(-1) spectral splitting for the two methyl groups at the vapor/dimethyl sulfoxide (DMSO, (CH(3))(2)SO) liquid interface as well as in the determination of their orientational angles.

The spatial distribution of bacteria in bulk soil has been well studied, but little is known about the bacterial biogeography in the rhizosphere of crops. Here, we investigated bacterial distribution in bulk soil, loosely-and tightly-bound soils, from wheat fields distributed across 800,000 km 2 of the North China Plain. Bacterial community composition differed dramatically among bulk and rhizospheric soils, and bacterial diversity decreased with the root proximity. Soil pH correlated with bacterial community composition and diversity in three compartments. Bacterial community in tightly bound soil formed a hub-based network topology with higher transitivity and greater number of central nodes compared with loosely bound and bulk soils, potentially as a result of more direct ecological interactions between the members of the tightly bound soil compartment. Bulk and rhizospheric soils maintained similar compositional distance decay patterns (with equal decay rates), but distinct phylogenetic distance decay patterns (with steeper slope of tightly bound soil). Geographical distance described a relatively greater proportion of bacterial spatial distribution in tightly bound soil, compared with loosely bound soil and bulk soil. Deterministic processes dominated the assemblage of bacterial communities in all soil compartments, while phylogenetic clustering was weaker in tightly bound soil. Taken together, our results suggest distinct bacterial network structure and distribution patterns among bulk soil, loosely bound soil and tightly bound rhizospheric soil, which could possibly result in potential functional differentiation.