Structural Analyses of the Ribosome by Chemical Modification Methods - Biophysical Approaches to Translational Control of Gene Expression - page 70

reactions can be performed anywhere along an RNA, thus enabling the structure of
any RNA to be examined, regardless of length. This represented a significant
advancement, allowing for secondary and some tertiary structure analyses to be
performed on RNAs which were too large for crystallographic studies. Additionally,
examination of the chemical protection patterns of sequences conserved between
species, differences in folding, and secondary structure were easily demonstrated.

4.2 Base-Speci fi c Chemicals

4.2.1 Introduction

A variety of chemicals were employed to modify RNAs during the early period of
chemically based structural analysis (Ehresmann et al. 1987 ; Weeks 2010 ) . Each
reagent was either base-specific or was intrinsically constrained by RNA structural
elements, providing specificity. Most commonly, a series of chemicals were used to
probe the same stretch of RNA, with each chemical providing different information.
The resulting data were combined to give a series of potential structures or to fur-
ther flesh out existing structural data. These chemicals and their reactions with
RNAs are shown in Fig. 4.1 .

4.2.2 Dimethyl Sulfate

DMS was one of the initial chemicals used to probe the paired and stacked nature of
bases for structure determination (Peattie and Gilbert 1980 ) . With regard to
specificity, it can methylate the N-7 position of unpaired guanine, the N-3 position
of unpaired cytosine, and the N-1 position of unpaired adenine. The methylated
guanine is reactive to aniline cleavage, thus mainly of use in the end-labeled, strand
scission method. An unreactive guanine indicates the presence of noncanonical
base-pairing or Mg ++ coordination. While the methylated cytosine requires further
chemical treatment by hydrazine to be detected using strand scission, it can be
detected directly using primer extension. The methylated adenines are only
detectable using a primer extension-based method.

4.2.3 Diethylpyrocarbonate

DEPC was initially used in determining the secondary structure of yeast tRNA
(Peattie and Gilbert 1980 ). It reacts with N-7 of adenine to create a site available for
cleavage by aniline in the strand scission method. Unfortunately, all adenines, paired
or unpaired, become unreactive in helices due to the higher sensitivity DEPC has for