Biomedical subjects

Jason A Mears

Publications and source records attributed to Jason A Mears.

5 recordsLinked to original sources

PubMed2006

A structural model for the large subunit of the mammalian mitochondrial ribosome.

Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1A resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.

Animals↗

PubMed2005

Dnm1 forms spirals that are structurally tailored to fit mitochondria.

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

Dimerization↗

PubMed2004

Using cross-links to study ribosomal dynamics.

After publications of 3-D models of a static ribosome and its large and small subunits, one of the next tasks is to recognize movable ribosomal elements responsible for mechanical shifts during protein synthesis. Statistic analysis of available cross-linking data allowed us to reveal three well separated groups of motions in the ribosome: I, mean magnitude of 10 A; II, most abundant, centered at 20 A and of wide dispersion, and III, sparsely populated, with large distances up to 95 A. The last group, III, comprises elements, like the L7/12-stalk and the L1- protuberance, that adopt different positions in crystallographic or electron micrographic structures, and neighboring hairpins 88 and 89, indicating mobility. We demonstrate that the cross-linking method can be applied to study ribosomal dynamics, including large-scale functional movements and, in particular, to estimate which structures participate in molecular switches.

Cross-Linking Reagents↗

PubMed2003

A structural model for the assembly of the 30S subunit of the ribosome.

The order in which proteins bind to 16S rRNA, the assembly map, was determined by Nomura and co-workers in the early 1970s. The assembly map shows the dependencies of binding of successive proteins but fails to address the relationship of these dependencies to the three-dimensional folding of the ribosome. Here, using molecular mechanics techniques, we rationalize the order of protein binding in terms of ribosomal folding. We determined the specific contacts between the ribosomal proteins and 16S rRNA from a crystal structure of the 30S subunit (1FJG). We then used these contacts as restraints in a rigid body Monte-Carlo simulation with reduced-representation models of the RNA and proteins. Proteins were added sequentially to the RNA in the order that they appear in the assembly map. Our results show that proteins nucleate the folding of the head, platform, and body domains, but they do not strongly restrict the orientations of the domains relative to one another. We also examined the contributions of individual proteins to the formation of binding sites for sequential proteins in the assembly process. Binding sites for the primary binding proteins are generally more ordered in the naked RNA than those for other proteins. Furthermore, we examined one pathway in the assembly map and found that the addition of early binding proteins helps to organize the RNA around the binding sites of proteins that bind later. It appears that the order of assembly depends on the degree of pre-organization of each protein's binding site at a given stage of assembly, and the impact that the binding of each protein has on the organization of the remaining unoccupied binding sites.

Binding Sites↗

PubMed2002

Modeling a minimal ribosome based on comparative sequence analysis.

We have determined the three-dimensional organization of ribosomal RNAs and proteins essential for minimal ribosome function. Comparative sequence analysis identifies regions of the ribosome that have been evolutionarily conserved, and the spatial organization of conserved domains is determined by mapping these onto structures of the 30S and 50S subunits determined by X-ray crystallography. Several functional domains of the ribosome are conserved in their three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplast ribosomes. In contrast, other regions from both subunits have shifted their position in three-dimensional space during evolution, including the L11 binding domain and the alpha-sarcin-ricin loop (SRL). We examined conserved bridge interactions between the two ribosomal subunits, giving an indication of which contacts are more significant. The tRNA contacts that are conserved were also determined, highlighting functional interactions as the tRNA moves through the ribosome during protein synthesis. To augment these studies of a large collection of comparative structural models sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans mitochondrial rRNA is considered individually because it is among the smallest rRNA sequences known. The C.elegans model supports the large collection of comparative structure models while providing insight into the evolution of mitochondrial ribosomes.

Animals↗