Nuclear Overhauser experiments at 500 MHz on the downfield proton spectra of 5S ribonucleic acid and its complex with ribosomal protein L25

Abstract

The downfield (9-15 ppm) proton spectrum of Escherichia coli 5S RNA has been examined at 500 MHz by using nuclear Overhauser methods. The data confirm the existence of the terminal and procaryotic loop helices within the molecule [Fox, G. E., & Woese, C. R. (1975) Nature (London) 256, 505-506]. Very little stable, double-helical structure is detectable in the third loop of the molecule, the one comprising bases 12-68. The downfield spectrum of 5S RNA is perturbed in a highly specific manner upon addition of protein L25 to the system. The changes seen strongly suggest that the binding site for L25 on 5S RNA includes the procaryotic loop helix, but not the terminal stem helix. Similar complexes formed between L25 and the 5S RNA fragment consisting of bases 1-11, 69-87, and 89-120 show exactly the same spectral alterations. A number of downfield resonances appear in the spectra of these complexes which have no counterparts in the free RNA, suggesting the stabilization of new RNA structures by the protein. There are some indications of protein-nucleic acid nuclear Overhauser effects.

Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.

“…In addition, the guanosine-81 and cytosine-93 residues are protected from cleavage by the double-strand-specific cobra venom endonuclease on addition of L25 to 5S rRNA (29), further implicating helix IV as part of the binding site. Moreover, analysis by NMR spectroscopy of the downfield proton spectra of 5S rRNA taking advantage of nuclear Overhauser effects reveals perturbations in the presence of L25 that also suggest a binding site for the protein in helix IV and exclude the molecular stalk (helix I) as part of the site (30). We find that L25 protects residues 72-109 from digestion by a-sarcin (Fig.…”

Exploration of the L18 binding site on 5S RNA by deletion mutagenesis

“…Ribosomal protein bL18 from Bacillus stearothermophilus was purified from 70S ribosomes by chromatography on carboxymethyl cellulose and by HPLC on SP5W columns in urea-containing buffers (15). Ribosomal protein L25 from E. coli was prepared as described elsewhere (7). Enzymes and Reagents.…”

NMR Spectroscopy of RNA

“…Furthermore, NOE contacts between the imino protons of neighboring base pairs are observable and facilitate the sequential assignment of the imino proton signals (Table 2). With this information, it has been possible to derive secondary structure models of tRNA molecules26–28 and 5S rRNA 29–31…”

“…The most straightforward method is the observation of chemical‐shift changes of the RNA upon gradual addition of the ligand—a method called chemical‐shift mapping. Probably the first application of this method to RNA was reported by Kime and Moore in 198331 in an investigation of the binding of the ribosomal protein L25 to 5S rRNA from E. coli. In their pioneering work, they used changes in the 1D 1 H spectra of the imino proton region of 5S rRNA upon addition of rL25 to identify nucleotides in the E‐loop region of 5S rRNA as the possible protein binding site.…”