The role of small proteins, peptides and dual function transcripts in cyanobacteria

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Genes possessing very short reading frames (smORFs) and dual function transcripts are only to a very limited number identified during the standard annotation of microbial genomes. Based on the comparative analysis of transcriptomic and genomic datasets, we predicted 45 smORFs encoding small proteins with ≤50 and 293 with ≤80 amino acids in the photosynthetic model organism Synechocystis sp. PCC 6803. The existence of six small proteins ≤50 and of four >50 but ≤70 amino acids was shown immunologically after epitope tagging and by peptidomics. Six of these small proteins were selected for closer analysis and then we focused on two of them, AtpΘ and NblD. Through the characterization of mutants and the analysis of interacting proteins by mass spectrometry, AtpΘ was characterized as a previously unknown inhibitory factor of ATP synthase. AtpΘ is a 48 amino acids small protein that is widely conserved throughout the cyanobacterial phylum, specifically expressed during low energy conditions such as during the night, binds to the α and γ subunits of ATP synthase and inhibits its ATPase activity if the electrochemical gradient collapses. Hence, AtpΘ resembles the alpha-helical peptide inhibitors of ATP synthases characterized in eukaryotic mitochondria. With this, it constitutes a remarkable instance of convergent evolution and adds another functionality to the growing class of small proteins in bacteria. NblD is a 66 amino acids cysteine-rich small protein that becomes specifically expressed during nitrogen starvation. Homologs of NblD exist in all cyanobacteria that use phycobilisomes for photosynthetic light harvesting but not in genera that use alternative light-harvesting mechanisms. We found that NblD binds to the β phycocyanin subunit where it plays a crucial role in the coordinated dismantling of phycobilisomes under nitrogen starvation.During the second funding period we will in collaboration with the Z2 unit use the full power of Ribo-seq and Ribo-TIS/TTS for profiling of translation initiation and termination sites in Synechocystis as well as perform Ribo-seq-based analysis of translatome changes under 4 different conditions. We will then focus on the functional in-depth characterization of AtpΘ and NblD and unravel the involved molecular, structural and regulatory details. We will benefit from the interaction with both the Z1 and the Z2 unit and with several other groups of the consortium as well as contribute as collaborator to several selected other projects.