eTopics

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eTopic 8.1 Discovering the mRNA Ribosome-Binding Site

In an elegant yet simple experiment, Joan Steitz was able to provide evidence of a ribosome-binding site on mRNA. Steitz took a radioactive, 30-nt, 32P-labeled RNA fragment from the 5′ end of an mRNA molecule and added it to ribosomes. The 5′ end of the mRNA is where the initiation sequences should be. Steitz then treated the ribosome initiation complex with colicin E3, a toxin made by some bacteria as a defense against others. Colicin E3 cleaved the large 16S rRNA molecule once, about 50 nt from the 3′ end, to yield a small, manageable piece that would easily enter a gel. The ribosomal proteins were then peeled away using the detergent SDS (sodium dodecyl sulfate), and the mixture was subjected to electrophoresis through a polyacrylamide gel to separate different RNA bands by size. The gel was dried and placed onto X-ray film to visualize the RNA bands. SDS does not disrupt RNA-RNA interactions; consequently, if the 3′¢ end of 16S rRNA oriented the mRNA message for translation by binding to the 5′¢ end of the mRNA, a 50-nt + 30-nt (rRNA-32P-labeled mRNA fragment) hybrid molecule would exist and, on the basis of its size, travel to a specific position in the gel. When the film was developed, the radioactive band was exactly where Steitz predicted a 50 nt–30 nt hybrid should be (Fig. 1).

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Figure 1  Experiment that revealed the Shine-Dalgarno ribosome-binding site in E. coli.  

Steitz, Joan A., and Karen Jakes. 1975. How ribosomes select initiator regions in mRNA: Base pair formation between the 3′ terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proceedings of the National Academy of Sciences USA. 72:4734–4738.