Modular and Versatile Trans-Encoded Genetic Switches

Current bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans-encoded and based on tRNA-mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA.     

BIOLOGICAL FUNCTIONS OF MODIFIED NUCLEOTIDES IN tRNA MOLECULES (Ⅲ)——SYNTHESIS AND BIOLOGICAL ACTIVITY OF ANALOGS OF YEAST ALANYL tRNA WITH m~1I_(37) REPLACED BY A, G OR C

A thirty-nine nucleotide fragment (nucleotides 38—76) of yeast alanyl tRNA was prepared by using calf spleen phosphodiesterase to delete C_(36)m~1I_(37) from the 3′-half molecule of this tRNA under controlled conditions. Analogs of yeast alanyl tRNA with A, G or C instead of m~1I_(37) were synthesized and their biological activities determined. The results indicated that the aminoacylation activity of these analogs was not affected in the rat liver aminoacyl-tRNA synthetase system, compared with natural yeast alanyl tRNA. However, the incorporation activity (i. e. the activity of transferring alanine into proteins) of these analogs was significantly reduced to 20—30% of that of natural yeast alanyl tRNA in the rabbit reticulocyte lysate cell-free protein synthesizing system.     

Visualizing the dual space of biological molecules

An important part of protein structure characterization is the determination of excluded space such as fissures in contact interfaces, pores, inaccessible cavities, and catalytic pockets. We introduce a general tessellation method for visualizing the dual space around, within, and between biological molecules. Using Delaunay triangulation, a three-dimensional graph is constructed to provide a displayable discretization of the continuous volume. This graph structure is also used to compare the dual space of a system in two different states. Tessellator, a cross-platform implementation of the algorithm, is used to analyze the cavities within myoglobin, the protein-RNA docking interface between aspartyl-tRNA synthetase and tRNA^Asp, and the ammonia channel in the hisH–hisF complex of imidazole glycerol phosphate synthase.     

A Covalent Approach for Site‐Specific RNA Labeling in Mammalian Cells

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site‐ and sequence‐specific covalent labeling of RNAs in mammalian cells by using tRNA^Ile2‐agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide‐bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site‐specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.