The<i>Escherichia coli</i>ibpA thermometer is comprised of stable and unstable structural elements

Abstract

Translation of many small heat shock genes in alpha- and gamma-proteobacteria is controlled by the ROSE (Repression Of heat Shock gene Expression) element, a thermo-responsive RNA structure in the 5'-untranslated region. ROSE(ibpA) regulates translation of the Escherichia coli ibpA gene coding for an inclusion body-associated protein. We present first structural insights into a full-length ROSE element by examining the temperature-induced conformational changes of ROSE(ibpA) using detailed enzymatic and lead probing experiments between 20 and 50 degrees C. The initial two hairpins are stable at all temperatures tested and might assist in proper folding of the third temperature-responsive stem-loop structure, which restricts access to the Shine-Dalgarno sequence at temperatures below 35 degrees C. Toeprinting (primer extension inhibition) experiments show that binding of the 30S ribosome to ROSE(ibpA) is enhanced at high temperatures. In contrast to other ROSE-like elements, the final hairpin is rather short. Single point mutations result in alternative structures with positive or negative effects on translation efficiency. Our study demonstrates how the combination of stable and unstable modules controls translation efficiency in a complete RNA thermometer.