Ribosome recycling factor (RRF) is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of RRF resembles a tRNA shape, with an architecturally different flexibility compared with tRNA, but its structure-and-function relationships are unknown. We here found that an RRF variant defective in ribosome binding regains the binding capacity through 20 independent secondary changes occurring in three topologically distinct regions of RRF. Because two of these regions are equivalent to the tip of the anticodon stem and the upper surface of the acceptor stem of tRNA, RRF may interact with the ribosome in a way similar to tRNA, spanning 30S and 50S subunits, to exert its action for splitting the ribosome.

The 5′ untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused. Previous studies have identified two domains of this 5′ UTR that together are responsible for its stabilizing effect. One is a 5′-terminal stem-loop. The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA. Here we report a detailed investigation of the function of these two stabilizing elements. Our data indicate that mRNA protection by a 5′ stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation. Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5′ UTR segment by bound ribosomes. Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5′ UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region. Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5′ stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent. These findings imply that the ompA 5′ UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation.