From equilibrium to kinetic footprinting of RNA structure

Abstract

RNA is a central molecule in the transmission, processing, and translation of genetic information. Although the critical role of RNA in cellular processes has long been recognized, the discoveries of autocatalytic 1 and catalytic processes 2,3 that are RNA-based have spurred even more interest in the chemistry and biology of RNA systems. The unique three dimensional structures adopted by various RNAs determine their activity and thus an understanding of structure is key to a full appreciation of their biological roles. Advances in high-resolution structure determination have provided insight into the organization and function of increasingly complex biochemical entities involved in RNA processing culminating in the recent structure of the large ribosomal subunit. Despite these impressive achievements, structural analysis by X-ray or NMR techniques remains an arduous and sometimes capricious endeavour. There is thus a need for simple chemical probes to provide structural information in the absence of higher resolution data and to complement those data where they have been obtained. Chemical tools for the analysis of nucleic acid structure were largely developed for use in characterization of DNA or protein–DNA complexes. Because RNA differs from DNA only by the presence of a 2 -hydroxy group, methodologies developed for the study of DNA systems have proven useful in the study of RNA structure and function as well. Many of these tools are reagents which react with some nucleic acid functionality in a specific fashion and render the phosphodiester backbone labile to cleavage. RNAs of different sizes, radio-labeled at the 5 or 3 ends with P, may be separated at nucleotide resolution by denaturing polyacrylamide gel electrophoresis (PAGE) and then visualized by exposure of the gel to either X-ray film or another imaging surface. Nucleic acid fragments appear as bands in the gel; smaller fragments migrate faster than larger ones and the exact position in a particular sequence may be ascertained by comparison to a “ladder” of bands generated by partial alkaline hydrolysis, base-specific chemical sequencing, enzymatic sequencing, or base-specific enzymatic cleavage (the enzyme RNaseT1, for example, cleaves RNA sequences only after guanosine residues). When a chemical reagent is used to induce cleavage of the RNA backbone, the presence of a band on a gel indicates that the targeted functionality was accessible to the modifying reagent while the absence of a band indicates that the functionality was inaccessible due to the formation of some kind of interor intra-molecular structure. Regions of decreased band intensity spanning one or more nucleotides are referred to as footprints and this type of experiment, known as protection footprinting, has been of immense power in the analysis of structure in nucleic acid systems (Fig. 1; interference footprinting involves pre-treatment of a nucleic acid with a modifying reagent in order to determine which modifications block structure formation). A large group of reagents have been used in protection footprinting experiments. Gilbert and co-workers showed that dimethyl sulfate (DMS), which reacts with N7 of guanosine and N3 of adenosine, can be used to detect contacts to these positions. Diethyl pyrocarbonate† modification of DNA has been used as a probe of contacts to the N7 positions of G and A while osmium tetraoxide (OsO4) and potassium permanganate (KMnO4), which react with the 5,6 bond of the thymidine base, have been used as protection probes of changes in DNA helix parameters. Protection footprinting has typically been used to compare two different states in a system; for example, an unbound nucleic acid can be compared to a protein-bound nucleic acid or an unstructured nucleic acid can be compared to a structured or folded nucleic acid (Fig. 1). There have been a host of protection footprinting studies of protein–DNA systems ranging Fig. 1 Protection footprinting as a probe of nucleic acid structure. a) Formation of higher order structure in nucleic acids by protein–nucleic acid association (top) or folding of a nucleic acid (bottom). The structure of the complexed or folded molecule may be probed by treatment with a nucleic acid modifying agent which initiates strand cleavage. Arrows represent modification of the nucleic acid with such a reagent and shading denotes protection from modification; b) PAGE analysis of P radio-labeled fragments obtained in the protection experiment. Comparison is made of the cleavage patterns obtained for the unbound or unstructured state (A) with the bound or structured state (B). Areas of reduced band intensity are referred to as footprints and represent the protection from chemical modification resulting from protein binding or higher order structure formation.