The conformation of the Escherichia coli 10Sa RNA
(tmRNA) in solution was investigated using chemical and
enzymatic probes. Single- and double-stranded domains were
identified by hydrolysis of tmRNA in imidazole buffer and
by lead(II)-induced cleavages. Ribonucleases T1
and S1 were used to map unpaired nucleotides
and ribonuclease V1 was used to identify paired
bases or stacked nucleotides. Specific atomic positions
of bases were probed with dimethylsulfate, a carbodiimide,
and diethylpyrocarbonate. Covariations, identified by sequence
alignment with nine other tmRNA sequences, suggest the
presence of several tertiary interactions, including pseudoknots.
Temperature-gradient gel electrophoresis experiments showed
structural transitions of tmRNA starting around 40°C,
and enzymatic probing performed at selected temperatures
revealed the progressive melting of several predicted interactions.
Based on these data, a secondary structure is proposed,
containing two stems, four stem-loops, four pseudoknots,
and an unstable structural domain, some connected by single-stranded
A-rich sequence stretches. A tRNA-like domain, including
an already reported acceptor branch, is supported by the
probing data. A second structural domain encompasses the
coding sequence, which extends from the top of one stem-loop
to the top of another, with a 7-nt single-stranded stretch
between. A third structural module containing pseudoknots
connects and probably orients the tRNA-like domain and
the coding sequence. Several discrepancies between the
probing data and the phylogeny suggest that E. coli
tmRNA undergoes a conformational change.