Two Approaches to the Study of the Mechanism of the Transition Process from Initiation to Elongation in T7 Rna Polymerase

Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. However, the mechanism of the instability of abortive cycling in RNA polymerase is not been well understood. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies that reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes, we now propose that collapse contributes to the release of abortive products in the presence of all four NTPs. Specifically, we propose that stabilizing initially transcribing complexes against downstream bubble collapse will allow these complexes to escape better to full run elongation. This study will provide important mechanistic insight, but will also be valuable for the production of high quantities of RNA from highly abortive DNA sequences. We have recently proposed a new model for the transition from initiation to elongation in T7 RNA polymerase. DNA-to-DNA FRET measurements have allowed mapping the changes that occur before promoter release. To observe the movements of the rotating domain after promoter release requires protein labeling. We are developing a new technique of affinity directed site specific labeling of the protein. We have also begun to explore an intein mediated strategy for direct labeling of T7 RNA polymerase

Effects of Nanoscale Confinement on the Functionality of Nucleic Acids for Future Applications in Nanomedicine

The facile self-assembly and nanomanipulation of nucleic acids hold great promise in the design of innovative, programmable materials, with applications ranging from biosensing to cellular targeting and drug delivery. Little is known, however, of the effects of confinement on biochemical reactions within such systems, in which the level of packing and crowding is similar to that of intracellular environments. In this review article, we outline novel, unexpected properties of nucleic acids that arise from nanoscale confinement, as mainly revealed by atomic force and electron microscopy, electrochemistry, fluorescence spectroscopy, and gel electrophoresis. We review selected scientific studies over the last decade that describe the novel behavior of nanoconfined nucleic acids with respect to hybridization, denaturation, conformation, stability, and enzyme accessibility. The nanoscale systems discussed include self-assembled, water-soluble, DNA or RNA nanostructures, ranging in width from a few to several tens of nm; gold nanoparticles coated with DNA monolayers; and self-assembled monolayers of DNA, from a few to several hundreds of bp in length. These studies reveal that the functionality of nucleic acid-based nanosystems is highly dependent upon the local density, molecular flexibility and network of weak interactions between adjacent molecules. These factors significantly affect steric hindrance, molecular crowding and hydration, which in turn control nucleic acid hybridization, denaturation, conformation, and enzyme accessibility. The findings discussed in this review article demonstrate that nucleic acids function in a qualitatively different manner within nanostructured systems, and suggest that these novel properties, if better understood, will enable the development of powerful molecular tools for nanomedicine

Ribosome-Templated Azide–Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry

Over half of all antibiotics target the bacterial ribosomenature’s complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (<b>1</b>), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either <i>E. coli</i> 70S ribosomes or 50S subunits with macrolide-functionalized azide <b>2</b> and 3-ethynylaniline (<b>3</b>) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide <b>2</b> and five alkynes) and ultimately to a 16-component reaction (i.e., azide <b>2</b> and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the <i>anti</i> (1,4)-regioisomers as revealed by measured <i>K</i>_d values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed <b>2</b> and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (<b>1</b>)