Redox switch protein Hsp33 has a novel zinc-dependent DNA binding function under cold stress in Escherichia coli

The heat shock protein 33 (Hsp33), a redox-regulated molecular chaperone, protects Escherichia coli from H 2 O 2 and heat-induced stress. Although the function of oxidized Hsp33 has been studied extensively, the role of zinc-bound Hsp33 requires further investigation. This study reveals the indispensable functions of zinc-bound Hsp33 in nucleic acid binding and cold tolerance. We showed that recombinant zinc-bound Hsp33 protein binds to single- and double-stranded DNA, along with various nucleic acids, including luciferase mRNA and E. coli total mRNA. Moreover, the interaction between zinc ions and the zinc-binding domain plays a key role in the interaction between Hsp33 and DNA or RNA. To investigate the DNA binding of the Hsp33 protein and its physiological response to cold stress, we overexpressed Hsp33 in a cold-sensitive E. coli mutant strain. This treatment significantly enhanced cold-stress tolerance. Conversely, E. coli strains with mutations in the zinc-binding domain of Hsp33 did not show enhanced resistance to cold stress. These findings highlight the crucial role of the Hsp33 zinc-binding domain in response to cold stress. We also investigated the anti-terminal activity of Hsp33 and its mutations. Our findings demonstrate that Hsp33 overexpression enhances its anti-termination activity by dissolving the secondary stem-loop structure within the RNA termination region, thereby facilitating the expression of the chloramphenicol acetyltransferase gene. This is the first study to identify Hsp33 zinc-binding-dependent RNA chaperone activity during cold stress.