mRNA stabilization by the ompA 5′ untranslated region: Two protective elements hinder distinct pathways for mRNA degradation

Abstract

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.