Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.     

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.     

The conformational stability of ribosomal proteins L7 and L12 from E. coli. I. Circular dichroism study of the changes induced by ionic strength and solvents

Ribosomal proteins L₇ and L₁₂ are the only acidic proteins found on the 50S ribosomal subunit of Escherichia coli. The effect of ionic strength, helix-promoting solvents and denaturating agents on the conformation of these proteins has been studied. It has been established that the helicity of L₇ and L₁₂ proteins (approx. 45–50% α helix) can be increased to 60–70% when they are exposed to helix-promoting solvents such as methanol or ethanol in the presence of 0.1M salt. High ionic strength by itself was without any effect on the conformation of the proteins. However, the solvent, 2,2,2-trifluoroethanol increased the content of α helices up to 80% even in the absence of salt. Denaturating agents like urea (6M) or guanidine HCl (6M), decreased the content of the ordered structure below 20%. All conformational changes induced by salt or solvents were completely reversible and characterized by a broad transition showing a low degree of cooperativity. This might indicate the presence of discrete segments with variations in amino acid sequences and ordered structures with different stabilities.     

Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multivariate statistical analysis of ribosome electron micrographs. L and R lateral views of the 40 S subunit from HeLa cells

The morphology of the small (40 S) subunit of the eukaryotic ribosome from HeLa cells has been examined by single-particle averaging and multivariate image analysis applied to electron micrographs of negatively stained specimens. The use of multivariate image analysis allows different, independent components of the structural variation within the particles to be identified and separately studied. The largest component of variance for both lateral views (termed L and R) was the variation in the peripheral stain intensity. The second largest component of interparticle variation is due to changes in the particle appearance most likely associated with a change of orientation on the specimen film.Averages formed from particles falling within a small range of peripheral stain intensity allowed the changes in the projected structure to be studied as a function of local stain level. Visual observations of stain variation could be confirmed quantitatively.Significant differences were found between averages of particles in the L view and those in the R view. Multivariate image analysis of a mixed population of L and mirrored R particles showed that the differences consistently affect all particles. However, the R view increasingly resembles the L view as the overall level of stain is increased, in agreement with a model of partial stain immersion.     

Conformations and interactions of histone H2A (F2A2, ALK).

Conformational changes in histone H2A (ALK, F2A2, IIbl) as a function of ionic strength and pH have been followed using high resolution nuclear magnetic resonance (NMR), circular dichroism (CD), and infrared (ir). While change in pH from 3 to 7 (no added salt) causes little structural change, added salt induces the formation of both alpha helix (28 percent maximum) and intermolecular associates in the region of the molecule between 25 and 113. No beta structure was observed at high salt. By the use of different salts it was shown that the structural changes were due largely to nonspecific counterion screening by the added anion. Comparison of observed with simulated NMR spectra has led to the proposal that an ionic strength dependent equilibrium exists between largely unstructured coil molecules and fully structured and aggregated molecules. NMR spectra of H2A obtained in the presence of DNA showed that both the N- and C-terminal regions bind to DNA, i.e., not the portion of the chain that is involved in interhistone interactions.     

Efficient production of Clostridium botulinum exotoxin C3 in bacteria: a screening method to optimize production yields

Clostridium botulinum exoenzyme C3 is responsible for the inactivation of members of the Rho GTPase family that are implicated in actin-cytoskeleton reorganization. This property has been extensively used in the field to investigate the functionality of the Rho GTPases. However, systematic analysis of Rho GTPase functions requires large amounts of such inhibitors and consequently an optimization of the production yield of these proteins. Bacterial production of soluble proteins often requires a refolding step that noticeably affects the production yields and necessitates additional experiments to verify functional activity. This is particularly true for TAT-C3, the production yields of which are generally low. In this report, we describe a rapid and efficient method for the production of soluble C3 exoenzyme developed by screening a collection of bacterial strains. The recombinant C3 protein was fused to the TAT protein-transduction domain from HIV, to allow protein delivery into cells, and to a hexahistidine tag, that permitted purification by Nickel affinity chromatography. We have demonstrated the production of large amounts of soluble and functional protein using the bacterial strain AD494 (DE3)pLysS. This rapid and efficient method for the production of soluble C3 exoenzyme could also be useful for the production of other proteins with solubility problems.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryote chromatin. The conformation of histone H1.

Proton magnetic resonance, circular dichroism and other studies of whole and cleaved calf thymus histone H1 (formerly F1) reveal the presence of specific folded structures in the region approximately from residue 40--115. Ionic, hydrogen-bond and hydrophobic interactions all appear to contribute to the stability of the structure, which is predicted to contain alpha-helices in regions 42--55 and 58--75. No evidence was found for beta-structures, either inter or intramolecular, or for any structure formation outside the region 40--115. At 18 degrees C and a protein concentration of 2 mM the first-order exchange rate between random-coil and structured forms is slower than 80 s-1; at 40 degrees C the exchange rate is faster than 330 s-1.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL-15R alpha fusion proteins

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.