Sequence and Structure Determinants of Drosophila Hsp70 mRNA Translation: 5'-UTR Secondary Structure Specifically Inhibits Heat Shock Protein mRNA Translation

Abstract

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5′-untranslated region (5′-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5′-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5′-UTR was measured. Stem-loops of −11 kcal/ mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A −22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5′-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5′-UTR secondary structure, allowing only mRNAs with minimal 5′-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested.