Programmed Ribosomal Frameshifting Mediates Expression of the α-Carboxysome

• Published in: Journal of molecular biology 2016-01, Vol.428 (1), p.153-164
• Main Authors: Chaijarasphong, Thawatchai, Nichols, Robert J., Kortright, Kaitlyn E., Nixon, Charlotte F., Teng, Poh K., Oltrogge, Luke M., Savage, David F.
• Format: Article
• Language: English
• Subjects: bacterial microcompartments; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; carboxysome; co-translational regulation; CsoS2; Escherichia coli - genetics; Frameshifting, Ribosomal; Gene Expression Regulation, Bacterial; Halothiobacillus - genetics; Macromolecular Substances - metabolism; Protein Isoforms - biosynthesis; Protein Isoforms - genetics; Protein Multimerization
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2015.11.017

Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichiacoli, opening the door to detailed mechanistic analysis of the assembly process of this complex (more than 200MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a molecular function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent molecular weight incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess −1 frameshifting elements that lead to the production of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3′ secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes. [Display omitted] •The carboxysome protein CsoS2 is produced as two isoforms, each with an unknown role in the assembly of α-carboxysomes.•We show that a −1 programmed ribosomal frameshift is responsible for the production of CsoS2A.•We express CsoS2A and CsoS2B individually for the first time.•We conclude that only CsoS2B is necessary and sufficient for the structural formation of carboxysomes.