Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

Abstract

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.

DNA and RNA: NMR studies of conformations and dynamics in solution

“…This 5-8S RNA is normally bound to the ribosome, contains modified nucleotides and sequence homologies are observed along its approximately 160 nucleotide length. Temperature dependent studies of the imino protons demonstrate a thermally stable GC rich stem whose simplified imino proton spectrum at high temperature has been assigned by i-D NOE experiments (Lee & Marshall, 1986). Thus, it may be possible to identify and assign by NMR thermally stable segments of larger RNAs at high temperature.…”

“…An imino proton NMR study has been recently reported on intact 5-8S RNA in aqueous solution (Lee & Marshall, 1986). This 5-8S RNA is normally bound to the ribosome, contains modified nucleotides and sequence homologies are observed along its approximately 160 nucleotide length.…”

Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA

“…Yeast 5S RNA Does Not Possess a Uniquely Heat-Stable Secondary-Structural Segment. On the basis of a combination of proton homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz spectrum, we were able to identify and assign eight of the nine base pairs in the most thermally stable helical arm (rich in G-C pairs) of yeast 5.8S rRNA (Lee & Marshall, 1986a). However, it is obvious from Figure 2 that none of the proposed secondary helices of yeast 5S rRNA contain an extended sequence of consecutive G-C base pairs.…”

“…Strategies applied to RNA NMR spectral assignments include ring current prediction (Arter & Schmidt, 1976;Hurd & Reid, 1979a,b; Shulman et al, 1973); isolation of enzymatic cleavage fragments (Boyle et al, 1980;Kime & Moore, 1983; 0006-2960/87/0426-5534S01.50/0 Li & Marshall, 1986;Chen & Marshall, 1986;Li et al, 1987); spectral comparisons between RNAs of similar primary sequence (Rordorf et al, 1976;Hurd & Reid, 1979a;Chen & Marshall 1986); proton homonuclear NOE's; and change in salt concentration or temperature to induce differential shifts or intensity changes in selected resonances (Heerschap et al, 1982(Heerschap et al, , 1983aKime & Moore, 1983; Li & Marshall, 1986;Lee & Marshall, 1986a;Lee, 1986).…”

“…Proton NMR characterization of purified RNA fragments obtained via specific enzymatic cleavage (Kime & Moore, 1983;Kime et al, 1984;Li & Marshall, 1986;Chen & Marshall, 1986) offers a promising approach, provided that the fragments retain the structure of the intact 5S rRNA molecule. Differential temperature-induced chemical shifts have been exploited successfully by Lee and Marshall (1986a) to demonstrate the existence of the exceptionally thermally stable common G-C-rich arm in yeast 5.8S rRNA. in 5S rRNA is rendered difficult by the apparent lack of any uniquely thermally stable helical secondary-structural segment.…”