The ribosomal domain of the bacterial release factors

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Abstract
1 Polyclonal antibodies (pAb 1–73 and pAb 26–120) have been raised against both an N‐terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1–73), and a fragment lacking part of the N‐terminal domain (amino acids 26–120). 2 Only pAb 26–120 inhibited release‐factor‐dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C‐terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3 A monoclonal antibody against an epitope of the C‐terminal two thirds of the protein (mAb 74–120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4 A monoclonal antibody against an epitope of the N‐terminal fragment of L7/L12 (mAb 1–73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release‐factor‐mediated events. 5 The mAb 74–120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1–73, indicating that the body L7/L12 dimer, and in particular its C‐terminal domains, are important for release factor/ribosome interaction. 6 The two release factors have subtle differences in their binding domains with respect to L7/L12.