Secondary Structure Mapping of an RNA Ligand That Has High Affinity for the MetJ Repressor Protein and Interference Modification Analysis of the Protein-RNA Complex

Abstract

The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box.Several genes involved in the biosynthesis of methionine in Escherichia coli are transcriptionally regulated by the methionine repressor, MetJ (1). The basic interaction occurs between a homodimer of the 12-kDa MetJ repressor subunits and an 8-base pair sequence that constitutes a Met box (2). The Met box is a tandem repeat that occurs between two and five times in natural operators and to which additional repressor dimers bind in a cooperative manner (3). The protein has a ␤␣␣ topology where the ␤-strands from each subunit intertwine to form an anti-parallel ␤-ribbon, which lies in the major groove of the target DNA. Binding specificity is largely determined by hydrogen bonds between the side chains of lysine 23 and threonine 25 of the ␤-strands and 4 purine bases in each Met box. Many hydrogen bonds are also made between the protein and the DNA phosphate backbone, and there is evidence that the conformation of the backbone is also important for binding (4). Binding of the MetJ repressor to DNA is modulated by Sadenosylmethionine (AdoMet), 1 which markedly increases the affinity of MetJ for its target sequence. AdoMet and the DNA are bound on opposite faces of the protein, and binding of AdoMet does not significantly perturb the protein structure. The increased affinity of the holorepressor appears to be due to a long range electrostatic effect caused by the positively charged tertiary sulfur atom of AdoMet (5, 6).Over the last decade in vitro selection and amplification techniques have been developed to isolate tight binding nucleic acid ligands for a wide range of target molecules (7,8). RNA molecules in particular have been isolated for a diverse set of targets, including proteins (9), amino acids (10), and nucleotides (11). These RNA aptamers potentially have very widespread applications as lead compounds in therapeutic situations, e.g. RNA-based inhibitors of the type I human immunodeficiency virus (HIV-I) reverse transcriptase (12, 13) or as ligands in diagnostic kits and biosensors. Despite their importance we have relatively little structural information on the ways in which aptamers interact with protein targets. Recently, one of our laboratories reported the first x-ray crystal structure for an aptamer bound to the RNA bacteriophage MS2 coat protein (14). T...

The metal- and DNA-binding activities of Helicobacter pylori NikR

“…It is also possible that the HpNikR interaction is dictated in part by the structure of DNA, and it has been suggested that the palindromic sequence of the ureA promoter forms a hairpin structure [10]. In support of this hypothesis, in vitro selection of RNA apatamers revealed that the ribbon-helix-helix MetJ protein binds a consensus sequence with significant secondary structure [35].…”

Characterization of RNA aptamers that disrupt the RUNX1–CBFβ/DNA complex

“…In addition to the selected pool a number of negative control RNAs were also assayed. These were the naïve library used for the current selections, an individual aptamer from the same library but selected against the bacterial transcription factor MetJ (33) or an aptamer pool derived from an unrelated library and selected against the amyloid forming protein β2m (30). These controls allowed us to show that any effects were due to the selected region of the anti-RHD–CBFβ pool, and were independent of the fixed flanking regions.…”

HIV-1 Reverse Transcriptase-Pseudoknot RNA Aptamer Interaction Has a Binding Affinity in the Low Picomolar Range Coupled with High Specificity

“…Dephosphorylation of the in vitro transcribed RNA prior to end labeling was carried out according to standard procedures (31). The fluorescent-labeled 5Ј-hexachlorofluorescein (HEX) pseudoknot RNA was synthesized and high pressure liquid chromatography-purified as described previously (32). HEX phosphoramidite was obtained form Glen Research (Sterling, VA).…”