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

在微生物基因组的标准注释中，具有很短阅读框架(SmORF)和双重功能转录本的基因只有非常有限的数量。基于转录和基因组数据的比较分析，我们在光合作用模式生物聚球藻中预测了45个编码小蛋白的单核苷酸重复序列(≤50)和293个单核苷酸重复序列(≤80)。PCC 6803。抗原表位标记和多肽分析表明，存在6种小蛋白≤50和4种≤70氨基酸。我们选择了其中的六个小蛋白进行进一步的分析，然后我们重点研究了其中的两个，三磷酸腺苷Θ和NblD。通过对突变体的鉴定和相互作用蛋白的质谱分析，确定了三磷酸腺苷Θ是一种未知的三磷酸腺苷合成酶抑制因子。三磷酸腺苷Θ是一种由48个氨基酸组成的小蛋白，在整个蓝藻门中广泛保守，在夜间等低能量条件下特异表达，与三磷酸腺苷合成酶的α和γ亚基结合，并在电化学梯度崩溃时抑制其三磷酸腺苷酶的活性。因此，三磷酸腺苷Θ类似于真核细胞线粒体中的三磷酸腺苷合成酶的α-螺旋肽抑制剂。有了这一点，它构成了一个非凡的收敛进化的例子，并为细菌中日益增长的小蛋白类别增加了另一种功能。NblD是一种66个氨基酸的富含半胱氨酸的小蛋白，在氮饥饿期间变得特异表达。NblD的同源物存在于所有使用藻胆体进行光合作用捕光的蓝藻中，但不存在于使用替代捕光机制的属中。我们发现NblD结合在β藻蓝蛋白亚基上，在氮饥饿条件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，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的翻译起始和终止位点，以及在4种不同条件下基于Ribo-Seq的翻译体变化的分析。然后，我们将重点研究三磷酸腺苷Θ和NblD的功能特征，并揭开涉及的分子、结构和调控细节。我们将从与Z1和Z2单位以及与财团的其他几个小组的互动中受益，并作为协作者为几个选定的其他项目做出贡献。

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.