Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Fluid-solid coupling dynamic model for oscillatory growth of multicellular lumens.

The development of multicellular lumens involves the interplay of cell proliferation, oscillation, and fluid transport. In this paper, a fluid-solid coupling dynamic model is proposed to investigate the physical mechanisms underlying the oscillatory growth of lumens. On the basis of experimental observations, the periodic oscillation of a lumen is interpreted by the fracturing-healing mechanism of cell-cell contacts, which induces a hydraulic-controlled outward flow switch. This model reproduces the oscillations of lumen sizes, in agreement with the experimental results of Hydra regeneration. It is found that the overall change trend of the lumen volume is determined by the tissue development induced by cell proliferation and the fluid transport induced by the osmotic pressure, while the outward flow due to the fracturing of cell-cell contacts regulates the oscillatory volume and the stress level in an appropriate scope. This work not only deepens our understanding of biomechanical mechanisms under the development of fluid-containing lumens, but also provides a theoretical framework to rationalize the dynamics of lumen-like tissues.

NMR and ab initio studies of amination of ketenimine: direct evidence for a mechanism involving a vinylidenediamine as an intermediate.

High-level ab initio calculations were carried out in both gas phase and solvent (epsilon = 35.9) to estabilish that the amination of ketenimine proceeds via amine addition across the C=N bond rather than the C=C bond, followed by tautomerization to form amidine product. The HOMO of ketenimine is perpendicular to its molecular plane with the largest coefficient on C(beta), while the LUMO is in its molecular plane with the largest coefficient on C(alpha). Amination of ketenimine involves in-plane attack of amine nucleophile on C(alpha) (LUMO) of ketenimine. The labile vinylidenediamine intermediate trans-11 for the reaction of ketenimine 10 with n-butylamine was directly observed by means of low-temperature proton NMR spectra. The evidence confirms that the amination reaction is stepwise and proceeds via n-butylamine addition across the C=N bond of ketenimine 10 rather than the C=C bond, followed by a slower tautomerization of vinylidenediamine trans-11 to amidine 12. Even though the second step is much slower, the first step involving amine addition across the C=N bond is kinetic control. Surprisingly, in the reaction of 10 with n-BuNH(2), attack of n-BuNH(2) syn to the phenyl group on C(beta) of 10 is preferred, even though this produces a less stable product (trans-11); attack of n-BuNH(2) anti to phenyl group on C(beta) of 10 is lacking and results in serious nonbonding interactions between the two phenyls of the ketenimine, as they are pushed together in this transition state.