Advances in Engine Test Capabilities at the NASA Glenn Research Center's Propulsion Systems Laboratory

The Propulsion Systems Laboratory at the National Aeronautics and Space Administration (NASA) Glenn Research Center is one of the premier U.S. facilities for research on advanced aeropropulsion systems. The facility can simulate a wide range of altitude and Mach number conditions while supplying the aeropropulsion system with all the support services necessary to operate at those conditions. Test data are recorded on a combination of steady-state and highspeed data-acquisition systems. Recently a number of upgrades were made to the facility to meet demanding new requirements for the latest aeropropulsion concepts and to improve operational efficiency. Improvements were made to data-acquisition systems, facility and engine-control systems, test-condition simulation systems, video capture and display capabilities, and personnel training procedures. This paper discusses the facility s capabilities, recent upgrades, and planned future improvements

Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin. Biochemistry 66

Study of the nature of protein-rRNA complexes is a topical problem of modern molecular biology. Structural studies of rRNA-protein complexes are the most direct and precise method of analysis of these interactions. Because ribosomal proteins are most conservative during evolution, their complexes with specific RNA fragments provide an interesting model for studying RNA-protein interactions. Ribosomal protein S8 from E. coli plays a key role in assembling the small ribosomal subunit The major region of protein S8 binding on 16S rRNA was determined by partial hydrolysis with restric tion endonucleases The binding sites of protein S8 on 16S rRNA are similar in E. coli and T. thermophilus. It was shown that ACCELERATED PUBLICATION 0006 2979/01/6609 0948$25.00 ©2001 MAIK "Nauka / Interperiodica" * To whom correspondence should be addressed. Vol. 66, No. 9, 2001, pp. 948 953. Translated from Biokhimiya, Vol. 66, No. 9, 2001, pp. 1165 1171. Original Russian Text Copyright © 2001 Abstract-The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restrict ed by nucleotides 588 602 and 636 651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA frag ments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and syn thesized in preparative amounts in vitro using T7 RNA polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bac terial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37 nucleotide rRNA fragment from the same organism suitable for X ray analysis were obtained

Stability of the ‘L12 stalk’ in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria

The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called ‘L12 stalk’ on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the ‘L12 stalk’. The ‘L12 stalk’ plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent

Electron Microscopy of the Amphibian Model Systems Xenopus laevis and Ambystoma mexicanum

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy

Domain II of Thermus thermophilus ribosomal protein L1 hinders recognition of its mRNA

The two-domain ribosomal protein L1 has a dual function as a primary rRNA-binding ribosomal protein and as a translational repressor that binds its own mRNA. Here, we report the crystal structure of a complex between the isolated domain I of L1 from the bacterium Thermus thermophilus and a specific mRNA fragment from Methanoccocus vannielii. In parallel, we report kinetic characteristics measured for complexes formed by intact TthL1 and its domain I with the specific mRNA fragment. Although, there is a close similarity between the RNA-protein contact regions in both complexes, the association rate constant is higher in the case of the complex formed by the isolated domain I. This finding demonstrates that domain II hinders mRNA recognition by the intact TthL1

Archaeal ribosomal protein L1: the structure provides new insights into RNA binding of the L1 protein family

Background: L1 is an important primary rRNA-binding protein, as well as a translational repressor that binds mRNA. It was shown that L1 proteins from some bacteria and archaea are functionally interchangeable within the ribosome and in the repression of translation. The crystal structure of bacterial L1 from Thermus thermophilus (TthL1) has previously been determined. Results: We report here the first structure of a ribosomal protein from archaea, L1 from Methanococcus jannaschii (MjaL1). The overall shape of the two-domain molecule differs dramatically from that of its bacterial counterpart (TthL1) because of the different relative orientations of the domains. Two strictly conserved regions of the amino acid sequence, each belonging to one of the domains and positioned close to each other in the interdomain cavity of TthL1, are separated by about 25 Å in MjaL1 owing to a significant opening of the structure. These regions are structurally highly conserved and are proposed to be the specific RNA-binding sites. Conclusions: The unusually high RNA-binding affinity of MjaL1 might be explained by the exposure of its highly conserved regions. The open conformation of MjaL1 is strongly stabilized by nonconserved interdomain interactions and suggests that the closed conformations of L1 (as in TthL1) open upon RNA binding. Comparison of the two L1 protein structures reveals a high conformational variability of this ribosomal protein. Determination of the MjaL1 structure offers an additional variant for fitting the L1 protein into electron-density maps of the 50S ribosomal subunit

Domain II of Thermus thermophilus ribosomal protein L1 hinders recognition of its mRNA

The two-domain ribosomal protein L1 has a dual function as a primary rRNA-binding ribosomal protein and as a translational repressor that binds its own mRNA. Here, we report the crystal structure of a complex between the isolated domain I of L1 from the bacterium Thermus thermophilus and a specific mRNA fragment from Methanoccocus vannielii. In parallel, we report kinetic characteristics measured for complexes formed by intact TthL1 and its domain I with the specific mRNA fragment. Although, there is a close similarity between the RNA-protein contact regions in both complexes, the association rate constant is higher in the case of the complex formed by the isolated domain I. This finding demonstrates that domain II hinders mRNA recognition by the intact TthL1