Investigation of the secondary structure of Escherichia coli 5 S RNA by high-resolution nuclear magnetic resonance

Abstract

The base-pairing structure of Escherichia coli 5 S RNA has been studied by high-resolution proton nuclear magnetic resonance spectroscopy. In the first analysis, these experiments provide a direct measure of the A · U and G · C base-pair content. Measurements over a range of temperatures indicate that the number of base pairs decreases from 28 ± 2 at 30 °C to 16 to 18 in the 52 °C to 62 °C range. On heating to 72 °C, this number is further reduced to about eight. An analysis of the nuclear magnetic resonance spectra in terms of nearest-neighbor ring current field affects on the positions of the low-field proton resonances provides a highly detailed picture of the 5 S RNA in its different stages of melting. The nuclear magnetic resonance spectra of all possible helices containing three or more base-pairs were computed and used in conjunction with the observed spectral data, general thermodynamic considerations and observations on the spectra of other polynucleotides to identify the helical sections present in 5 S RNA at 50 to 60 °C. This analysis leads to a model that includes three helices containing ten, seven and four base-pairs, respectively. If the helices that are present at the high temperatures are assumed to be present at lower temperatures, then the base-pairing matrix shows that many other helices are automatically excluded from consideration. This information, along with the nuclear magnetic resonance difference spectrum (30 to 50 °C), is sufficient to identify the two helices that are added when the temperature is lowered to 30 °C. In this way, a model for the complete secondary structure of 5 S RNA at room temperature can be deduced, which is consistent with other chemicals and phyical data. None of the previously proposed models are consistent with the nuclear magnetic resonance data.