The Mechanisms of RNA SHAPE Chemistry

The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2′-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2′-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2′-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the E. coli ribosome, monitored the reactivity of 1490 nucleotides in 16S ribosomal RNA, and examined those nucleotides that were hyper-reactive towards SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2′-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3′-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias

Structural Studies of the Elongation Cycle of Protein Synthesis and Its Inhibition by Antibiotics

Protein synthesis takes place in four stages: initiation, elongation, termination and recycling. Elongation consists of delivery of a charged tRNA to the ribosome, peptide bond formation and translocation of the mRNA and tRNA, three steps forming a cyclic process, which is repeated for every amino acid added to a growing polypeptide chain. The result of the elongation cycle is the translation of the triplet genetic code contained in an mRNA, into the amino acid sequence of a protein. Since the 1960s it was appreciated that complex conformational changes must occur on the ribosome to accomplish translocation of mRNA and tRNA. Here I report x-ray crystallographic studies which shed light on how mRNA and tRNA are manipulated by the ribosome and mechanisms used by certain antibiotics to inhibit the elongation cycle of protein synthesis.Specifically research reported here argue that tRNA translocation is a stepwise process that involves discrete structural intermediates of the ribosome. I report structural evidence that the antibiotics clindamycin and chloramphenicol inhibit protein synthesis by interfering with aminoacyl-tRNA positioning in the peptidyl transferase center. Also, I hypothesize based on structural data and phylogenetic analysis, that the identity of the ribosomal RNA residues numbered in E. coli 752, 2055 and 2609 contribute to the specificity of many antibiotics for binding to bacterial, rather than archaeal or eukaryotic ribosomes

Mini-review Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code

a b s t r a c t Important viral and cellular gene products are regulated by stop codon readthrough and mRNA frameshifting, processes whereby the ribosome detours from the reading frame defined by three nucleotide codons after initiation of translation. In the last few years, rapid progress has been made in mechanistically characterizing both processes and also revealing that trans-acting factors play important regulatory roles in frameshifting. Here, we review recent biophysical studies that bring new molecular insights to stop codon readthrough and frameshifting. Lastly, we consider whether there may be common mechanistic themes in À1 and þ1 frameshifting based on recent X-ray crystal structures of þ1 frameshift-prone tRNAs bound to the ribosome

The Mechanisms of RNA SHAPE Chemistry

The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2′-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2′-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2′-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the <i>Escherichia coli</i> ribosome, monitored the reactivity of 1490 nucleotides in 16S rRNA, and examined those nucleotides that were hyper-reactive toward SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2′-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3′-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry