Identification and characterization of the mitochondrial RNA polymerase and transcription factor in the fission yeast Schizosaccharomyces pombe

We have characterized the mitochondrial transcription factor (Mtf1) and RNA polymerase (Rpo41) of Schizosaccharomyces pombe. Deletion mutants show Mtf1 or Rpo41 to be essential for cell growth, cell morphology and mitochondrial membrane potential. Overexpression of Mtf1 and Rpo41 can induce mitochondrial transcription. Mtf1 and Rpo41 can bind and transcribe mitochondrial promoters in vitro and the initiating nucleotides were the same in vivo and in vitro. Mtf1 is required for efficient transcription. We discuss the functional differences between Mtf1 and Rpo41 of S. pombe with Saccharomyces cerevisiae and higher organisms. In contrast to S. cerevisiae, the established model for mitochondrial transcription, S. pombe, a petite-negative yeast, resembles higher organisms that cannot tolerate the loss of mitochondrial function. The S. pombe and human mitochondrial genomes are similar in size and much smaller than that of S. cerevisiae. This is an important first step in the development of S. pombe as an alternative and complementary model system for molecular genetic and biochemical studies of mitochondrial transcription and mitochondrial–nuclear interactions. This is the first systematic study of the cellular function and biochemistry of Rpo41 and Mtf1 in S. pombe

On the problem of overlapping ω scans measured on thin films deposited on monocrystal substrates

CeO2thin films deposited on sapphire monocrystal substrates were used for an experimental study of the nature of extremely narrow overlapped maxima on X-ray diffraction ω scans. Full width at half-maximum (FWHM) values of such maxima typically reached the resolution function of the diffractometer. A comparative study of the influence of various diffractometer set-ups on the spectral characteristics of the X-ray beam in relation to the above-mentioned phenomenon was carried out. A surrounding (λmin− λmax) or (2θmin− 2θmax) of the strong substrate reflection was obtained, where a substrate contribution to an ω scan measured on thin-film reflection can be expected. Two possible origins of the narrow maxima are discussed: (a) a contribution of a part of the X-ray beam having λ ≠ λKαthat diffracts on a set of substrate crystallographic planes parallel to the thin-film crystallographic planes used for the ω-scan measurement; and (b) the presence in part of the thin film of a perfect monocrystal-like quality with practically no mosaicity. The principles of this approach and experimental procedure are reported, and on this basis it is possible to distinguish between the two possible origins of the narrow overlapped maxima. It is shown that under appropriate conditions, an extremely high quality CeO2thin film can be grown. The FWHM value of its ω scan can reach the value of diffractometer instrumental broadening obtained for a perfect monocrystal.</jats:p

Structure and possible mechanism of the CcbJ methyltransferase from<i>Streptomyces caelestis</i>

TheS-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ fromStreptomyces caelestiscatalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0 Å resolution, its native form at 2.7 Å resolution and its complex withS-adenosyl-L-homocysteine (SAH) at 2.9 Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ–SAM–substrate and CcbJ–SAH–product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H+ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.</jats:p