One gene, two proteins: coordinated production of a copper chaperone by differential transcript formation and translational frameshifting in Escherichia coli.

Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P1B -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P1B -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock.

Distinct functions of serial metal-binding domains in the Escherichia coli†P1 B -ATPase CopA.

P1 B -ATPases are among the most common resistance factors to metal-induced stress. Belonging to the superfamily of P-type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P1 B -ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N-terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal-binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein-internal pathway of copper and demonstrate the distal N-terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane-integral ion-binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P1 B -ATPases, which is governed by the length of the inter-domain linker.