Michael W. Clark

The L7/L12 stalk, a conserved feature of the prokaryotic ribosome, is attached to the large subunit through its N terminus

Abstract The L7/L12 stalk, a distinctive feature of the large ribosomal subunit from Escherichia coli , is shown to be a general morphological feature associated with large subunits from representative prokaryotes. In the gram-positive thermophile Bacillus stearothermophilus , the stalk consists of four copies of ribosomal proteins B.L13, the functional and amino acid sequence homologue of E. coli proteins L7/L12. That this protein comprises the stalk is shown by electron microscopy of 50 S subunits that have had B.L13 selectively removed, and by immune electron microscopic mapping of B.L13. In addition, a fragment consisting approximately of the N-terminal one-third of B.L13, known to compete with B.L13 for binding to B.L13-depleted 50 S cores, is shown to be located in the region of the stalk proximal to the body of the 50 S subunit, and the C-terminal two-thirds of B.L13 is shown to be located in the globular region of the stalk that is distal to the large subunit. Taken together these results suggest that in the isolated subunit at least three and probably all four chains of B.L13, and of the corresponding L7/L12 proteins in other prokaryotes, are arranged with the N termini of the proteins contacting the body of the 50 S subunit and with the C-terminal globular regions of the protein extending away from the subunit into the cytoplasm.

Helical arrays of Escherichia coli small ribosomal subunits produced in vitro.

Abstract In vitro conditions have been determined for obtaining ordered helical ribbons of small ribosomal subunits from Escherichia coli . These ribbons, suitable for study by three-dimensional reconstruction, are the first ordered arrays of ribosomes or ribosomal subunits to be produced in vitro . Although small ribosomal subunits remain in solution for extended periods (up to 6 months) during this procedure, their structural integrity, as assessed by acrylamide/agarose gel electrophoresis, by sucrose gradients, and by electron microscopy, is not significantly altered. Electron micrographs of ribbons of small subunits diffract to 60 A resolution. Optical diffraction patterns suggest that adjacent subunits within helical ribbons are related by a 2-fold screw parallel to the long axis of the ribbon and the helical repeat distance measured from electron micrographs is 220 A.

Mapping evolution with three dimensional ribosome structure

Summary Three dimensional ribosomal structure is highly conserved, even when organisms from different urkingdoms are compared. Hence it is extremely useful as a probe of distant evolutionary events. Using a parsimony analysis of ribosome structure, we have analyzed evolutionary relationships and obtained two results that stand in contrast with the standard evolutionary tree. First we find that the sulfur dependent archaebacteria, or eocytes, are topologically nearest neighbors to the eukaryotes rather than to the methanogens. We suggest that the depth of this division is appropriate for a separation at the urkingdom level. Secondly, our data indicate that the halobacteria have diverged from the eubacteria more recently than from any other known group of organisms. We interpret these results to indicate that the halobacteria are incorrectly placed in the archaebacteria, and should probably be included with the eubacteria into a larger group, the photocytes.

NanoArrays, the Next Generation Molecular Array Format for High Throughput Proteomics, Diagnostics and Drug Discover

Biomolecular array technology is an invaluable tool for rapid screening of nucleic acid mixtures. This approach has been tremendously successful both in its breadth of application and its commercial value. Entire genomes, including the human genome, have been screened by molecular array techniques. Arrays are a rapid and now routine method for analysis of expression patterns and their association with physiological states. Such a rapid, high throughput analysis of cellular expression is key to the expansion of our basic knowledge of the relationship between gene expression and organismal function, as well as to the understanding of the genetic component of disease states and the predisposition to disease. Despite the success of array technology for nucleic acid applications, a similar trend for proteins has not occurred. Due, in part, to the difficulties involved in production and labeling of proteins for solid state analysis, solid state arrays of proteins are not widely utilized. Protein function and interaction have been traditionally addressed by the combination of 2D gel electrophoretic separation and mass spectrometry to examine individual protein spots, a slow, tedious and expensive process. Another approach uses in vivo methods for examining protein-protein interactions by the two-hybrid system in yeast and mammalian cells [1]. Although the two-hybrid system has shown some success in finding new interaction between proteins in important cellular pathways, it is far more difficult, costly and time consuming than the solid state methods used for nucleic acids. BioForce Laboratory, Inc., has developed a solid state method for examining the interaction between a wide range of molecules in an array format. This technology involves several key technological innovations.

Evidence for a specific phylogenetic relationship between the methanogens Methanococcus thermolithotrophicus and Methanothermus fervidus, and the sulfur respiring Thermoproteus tenax

Summary Using three dimensional ribosome structure as a probe of evolutionary relationships, we have investigated the relationship between the methanogens and the sulfur dependent archaebacteria or eocytes. Specifically, we show that the large ribosomal subunit structures of Thermoproteus tenax, Methanothermus fervidus and Methanococcus thermolithotrophicus are closely related. We interpret this (rotting the tree with the data of others) to suggest that the methanogens are derived from a sulfur dependent organism that had ribosomes of the Thermoproteus tenax type.