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.     

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)     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Separation of cytoplasmic ribosomal proteins of Microsporum canis

The cytoplasmic ribosomal proteins of Microsporum canis were characterised in basic-acidic and basic-SDS two-dimensional polyacrylamide gel electrophoresis systems. The small subunit contained 28 proteins and the large subunit 38 proteins. The molecular weights of these proteins were in the range of 32,500 to 7600 and 48,000 to 11,000 in the small and large subunits, respectively. The 80S ribosomes showed 65 and 66 protein spots in basic-acidic and basic-SDS gel systems, respectively.     

Peripartum Cardiomyopathy Presenting With Ventricular Tachycardia: A Rare Presentation

A 25-year-old previously asymptomatic pregnant woman at 36 weeks'' gestation was noticed to have repetitive monomorphic ventricular tachycardia. A dilated left ventricle with moderately reduced systolic function was found on echocardiographic examination. This is a very rare presentation of peripartum cardiomyopathy (PPCMP) presenting with repetitive monomorphic ventricular tachycardia.     

A recently silenced, duplicate PgiC locus in Clarkia

Previous electrophoretic analysis showed that 17 diploid species of the wildflower Clarkia (Onagraceae) have two cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9), whereas 15 other diploid species have a single PGIC. Molecular studies revealed that the two isozymes in the former species are encoded by duplicate genes, PgiC1 and PgiC2, whereas the single isozyme in the latter is always encoded by PgiC1. Phylogenetic analysis of the nucleotide sequences implied that PgiC2 was silenced four times independently in the genus. Here we describe a psi PgiC2 from C. mildrediae, a species in which only PgiC1 is expressed. The discovery of the psi PgiC2 is significant because it confirms a formal prediction of the phylogenetic analysis. The psi PgiC2 includes 5,039 nucleotides corresponding to 18 of the 23 exons of PgiC, as well as the intervening introns and 3' nontranslated region. The absence of an increase of nucleotide substitutions in its "exons" suggests that the gene was silenced recently. The present study appears to be the first to establish that a specific duplicate gene locus regularly expressed in a group of related plant species has been silenced in one of them. The multiple independent silencings of PgiC2 suggest that it remained functional but inessential in ancestral lineages. We discuss the possibility that PgiC2 may have been preserved in these lineages by selection against mutants causing defective PGIC1- PGIC2 heterodimers.      

Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster.

Scanning transmission electron microscopic images of transfer RNAs reveal the molecular dimensions and compact morphology of these small macromolecules in unprecedented detail. Selective labeling of a sulfhydryl group on 2-thiocytidine enzymatically inserted at position 75 at the 3' end of yeast tRNA(Phe) with an undecagold cluster permits identification of this specific tRNA site by dark field STEM. Imaging of a single nucleotide at a defined location on the tRNA molecule should make it possible to localize in situ tRNAs at the A, P, and E sites of the ribosomal peptidyl transferase center, and in complexes of tRNA with enzymes and elongation factors. In addition, this approach may be used for the highly specific topographical mapping of other RNAs and/or biological macromolecular complexes.     

A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+-ATPase

The peripheral (catalytic) sector of vacuolar H+-ATPases contains five different polypeptides denoted as subunits A-E in order of decreasing molecular masses from 72 to 33 kDa. The gene encoding subunit B (57 kDa) of yeast vacuolar H+-ATPase was cloned on a 5-kilobase pair genomic DNA fragment and sequenced. Four open reading frames were identified in the sequenced DNA. One of them encodes a protein of 504 amino acids with a calculated Mr of 56,557. Hydropathy plot revealed no apparent transmembrane segments. Southern analysis demonstrated that a single gene encodes this polypeptide in the yeast genome. The amino acid sequence exhibits extensive identity with the homologous protein from the plant Arabidopsis (77%). This polypeptide also contains regions of homology with the alpha subunits of H+-ATPases from mitochondria, chloroplasts, and bacteria. However, less similarity was detected when it was compared with the beta subunits of those enzymes. The implication of these phenomena on the evolution of proton pumps is discussed.