Identification of ribosome–ligand interactions using cleavage reagents

Abstract

To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.

Probing Surface Structure of Sarcin Domain on Ribosomes of <i>Escherichia coli</i> by Complementary Oligo DNAs and Ribosome-Inactivating Protein

“…This evidence has also been substantiated by observations of shifting sedimentation found in the probe-bound 50S ( fig. 2) in this study, and by a previous report that the cleavage of bases of U2684 and G2686 that were located at further positions outside the sarcin domain can be triggered by an oligo DNA oligomer complementary to the sarcin loop (G2655-C2667) with phenanthroline-Cu(II) placed at the 5) [14].…”

“…We have been investigating the importance of the 3) side of the sarcin domain by compiling our results with other available data that probes the structure of the sarcin domain by different methods [14,17,21,22,24,25,30]. The position of each of the reactive nucleotides on the predicted secondary structure of the sarcin domain is summarized in table 1.…”

Probing surface structure of sarcin domain on ribosomes ofEscherichia coli by complementary oligo DNAs and ribosome-inactivating protein

“…Furthermore, the contact of these extra nucleotides was not observed on probe-free UV-irradiated 50S ribosomes, or on nonirradiated 50S ribosomes. 2) in this study, and by a previous report that the cleavage of bases of U2684 and G2686 that were located at further positions outside the sarcin domain can be triggered by an oligo DNA oligomer complementary to the sarcin loop (G2655-C2667) with phenanthroline-Cu(II) placed at the 5' [14]. These findings suggest that the regional structure of sarcin domain may be changed when the 3' side nucleotides of the domain are contacted by a complementary oligo DNA.…”

“…Thus,we propose that the tetraloop and the 3' side half of the stem (position 2655-2674, possibly extended to 2676) is exposed at the surface of ribosomes, while the nucleotides at 5' side half of the stem and possibly extending to two nucleotides on the loop (position 2646-2654) are buried in ribosome particles. g Data obtained from phenanthroline-Cu-mediated cleavage [ 14]. b Data from this study.…”

DNA and RNA Cleavage Mediated by Phenanthroline-Cuprous Oligonucleotides: From Properties to Applications

“…Firstly, tRNA-Phe was linked to phenanthroline via the introduction of 4-thiouridine in the tRNA sequence. This tRNA-Phe-OP caused cleavage mainly in the 23S rRNA when it was bound to precise sites in the ribosomal complex (namely, to P and E sites of 70S ribosome; Hill et al 1995). This phenanthroline-conjugate allowed an analysis of the neighborhood of the rRNA surrounding the tRNA binding site (Bullard et al , 1998.…”