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

具有非常短的阅读框（smORF）和双功能转录物的基因在微生物基因组的标准注释期间仅被鉴定到非常有限的数目。基于对转录组和基因组数据集的比较分析，我们预测了光合模式生物集胞藻PCC 6803中编码≤50个氨基酸的45个smORF和编码≤80个氨基酸的293个smORF。在表位标记和肽组学后，免疫学上显示存在6个小于50个氨基酸和4个大于50但小于70个氨基酸的小蛋白。选择这些小蛋白中的六种用于更仔细的分析，然后我们集中于其中的两种，AtpΘ和NblD。通过突变体的鉴定和质谱分析相互作用蛋白，AtpΘ被鉴定为以前未知的ATP合酶抑制因子。Atp 0是一种48个氨基酸的小蛋白，其在整个蓝藻门中广泛保守，在低能量条件下例如在夜间特异性表达，结合ATP合酶的α和γ亚基，并且如果电化学梯度崩溃则抑制其ATP酶活性。因此，AtpΘ类似于真核线粒体中表征的ATP酶的α-螺旋肽抑制剂。因此，它构成了趋同进化的一个显著实例，并为细菌中不断增长的小蛋白质类增加了另一种功能。NblD是一种富含66个氨基酸的半胱氨酸的小蛋白，其在氮饥饿期间特异性表达。NblD的同源物存在于所有使用藻胆体进行光合捕光的蓝藻中，但不存在于使用替代捕光机制的属中。我们发现NblD与β藻蓝蛋白亚基结合，在氮饥饿下藻胆体的协调分解中起着至关重要的作用。在第二个资助期内，我们将与Z2单位合作，利用Ribo-seq和Ribo-TIS/TTS的全部功能来分析集胞藻中的翻译起始和终止位点，并进行Ribo-seq-基于四种不同条件下的翻译组变化分析。然后，我们将专注于AtpΘ和NblD的功能深入表征，并解开所涉及的分子，结构和调控细节。我们将受益于与Z1和Z2单元以及该联盟其他几个小组的互动，并作为合作者为其他几个选定的项目做出贡献。

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.