No abstract

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than Ltyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-L-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

No abstract

Gas hydrate occurrence is one of the possible mechanisms invoked for iron sulfide formation.A high-resolution rock magnetic study was conducted in IODP Expedition 316 Hole C0008C located in the Megasplay Fault Zone of the Nankai Trough, offshore Japan. In this particular zone, no bottom simulating reflectors (BSR), indicating the base of the gas hydrate stability field, have been identified. Two hundred and eighteen Pleistocene samples were collected from 70 to 110 m CSF in order to document the changes in the concentration, grain size, and rock magnetic parameters of magnetic minerals, through the gas hydrate-bearing horizons. Two different populations of magnetic grains are recognized in the pseudosingle domain range. Three types of magnetic mineral assemblages are identified: iron oxides (magnetite), ferrimagnetic iron sulfides (greigite and pyrrhotite), and their mixture. Greigite and pyrrhotite are authigenic and constitute six layers, called IS1-IS6. IS1, IS3, IS4, and IS6 are associated with pore water anomalies, suggesting the occurrence of gas hydrates and anoxic conditions. IS2 and IS5 are probable gas hydrates horizons, although there is no independent data to confirm it. The remaining intervals are mainly composed of detrital iron oxides and paramagnetic iron sulfides. Two scenarios based on different diagenetic stages are proposed to explain the variations in the magnetic properties and mineralogy over the studied interval. The results suggest that rock magnetism appears useful to better constrain the gas hydrate distribution in Hole C0008C, and counterbalances the low resolution of pore water analyses and the absence of a BSR.