Structural Analyses of the Ribosome by Chemical Modification Methods - Biophysical Approaches to Translational Control of Gene Expression - page 69

that do not easily condense into complex three-dimensional shapes, single-stranded
RNA molecules do. Thus, if one were to expose a three-dimensionally complex RNA
molecule to a selection of RNA modifying chemicals, only those portions of the
RNA polymer exposed to the surface would react with the chemicals, while those
buried in the interior would not. Given the tools to unravel (denature) the RNA and
to detect and map the chemically modified RNA bases, it is possible to reconstruct
the original three-dimensional structure using the basic strategy described above.
Given the increasing importance of understanding the roles played by structurally
complex RNA molecules on the biology of the cell, the utility of this approach
becomes obvious.

4.1.2 Historical Background

The first major report detailing the use of chemical modification for mapping the
structure of an RNA was published in 1980 (Peattie and Gilbert 1980 ) . This work
used a radioactively labeled RNA (yeast tRNA-Phe), modified with chemicals uti-
lized in RNA sequencing methodologies employed at the time: dimethyl sulfate
(DMS) and diethylpyrocarbonate (DEPC), followed by strand scission using sodium
borohydride and aniline. The reactants were separated through large-format acryl-
amide gels, resolving the fragmented RNA species by molecular weight. Comparison
of the banding patterns relative to native, semi-denatured and denatured tRNAs,
enabled determination of whether or not a given base was likely involved in a base-
pairing or base stacking interactions. The results were largely in agreement with
crystal structures known at the time, but also enabled identification of additional
tertiary interactions in this tRNA that were previously unknown. Furthermore,
comments in the discussion section of the paper pointed to another innovation; by
chemically modifying the RNA and then purifying and labeling it, an RNA mole-
cule could be mapped in its native form without the need for renaturation. This
would prove to be especially useful when dealing with large RNAs, e.g., ribosomal
RNA (rRNA) or viral RNAs. Later groups (Stern et al. 1988 ) would expand the
chemicals used to modify the RNA to those where strand scission would no longer
be needed and which could interrogate a greater variety of bases.
The next major innovation in the field was introduced 3 years later (Hu and
Dahlberg 1983 ). In addition to introducing a larger variety of modifying chemicals
(discussed below), reverse transcription rather than end-labeling was employed to
produce fragments for analysis. In this report, RNAs were chemically modified,
denatured, and a DNA primer which had been radioactively labeled was annealed to
the modified RNA species. Primer extension employed avian myeoloblastosis virus
(AMV) reverse transcriptase, which produced strong stops 1 nucleotide 5¢ of each
modified base. The resulting cDNA products were separated through denaturing
acrylamide gels alongside a reverse transcriptase Sanger sequencing reaction. By
comparing the presence of bands in the chemically modified lanes against untreated
samples, base-pairing, stacking, and tertiary interaction status could be determined
for each nucleotide. Since primers can be designed for any sequence, extension