Poster abstracts
Poster number 95 submitted by Sezen Meydan
Programmed frameshifting generates a copper transporter and a copper chaperone from the same gene
F. Sezen Meydan (Center for Biomolecular Sciences, University of Illinois at Chicago, IL, USA), Dorota Klepacki, Subbulakshmi Karthikayen, Tonu Margus (Center for Biomolecular Sciences, University of Illinois at Chicago, IL, USA), Paul Thomas (Proteomics Center of Excellence, Northwestern University, Chicago, IL, USA), John Edward Jones, Yousuf Khan, Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA), Nora Vazquez-Laslop, Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago, IL, USA)
Abstract:
Ribosome profiling of E. coli cells showed an abrupt interruption of translation at the 70th codon of the copA gene that encodes a copper transporter. Our in vivo and in vitro experiments showed that the interruption of copA translation is due to a novel case of -1 programmed ribosomal frameshifting (PRF). The PRF within copA results in production of the full-size 834 amino acid-long transporter CopA and a 70 amino acid-long truncated product that we named CopA(Z) from a single gene. The PRF within copA gene occurs with a high efficiency nearing 50%. We have identified three main elements stimulating the -1 PRF of copA: (1) the presence of the slippery sequence CCC-AAA-G, (2) a downstream mRNA pseudoknot, and (3) the sequence of the CopA nascent chain in the ribosomal exit tunnel. Our phylogenetic analysis revealed that the stimulatory signals of -1 PRF in copA are evolutionarily conserved among a wide variety of bacteria, suggesting that the PRF is a common mechanism of regulating expression of this gene, which confers fitness advantage. Indeed, co-growth experiments demonstrated that cells carrying mutations that disrupt the slippery sequence, and hence lack the ability to produce CopA(Z) lose in competition with wt cells when challenged by toxic concentrations of copper. Since E. coli lacks an independent gene encoding for a copper chaperone, we suggest that this role is achieved by the PRF product CopA(Z) in E. coli and other bacteria. Strikingly, the same slippery sequence CCC-AAA-G is also observed in ATP7B gene encoding the CopA homolog in human. The dual luciferase reporter assays in HEK293T cells demonstrated that this sequence together with a predicted downstream pseudoknot is indeed able to induce -1 frameshifting with about 12% efficiency. Moreover, a similar arrangement of SS and a downstream pseudoknot is found in ATP7B homologs of higher primates. It is possible that in these organisms, -1 PRF could be involved in generating a copper transporter and a copper chaperone, with related but distinct functions in copper management. Our findings hence illuminate a unique translation regulation system crucial for the copper homeostasis in bacteria and likely in human and other eukaryotes.
Keywords: programmed frameshifting, ribosome profiling, pseudoknot