Role of small proteins in the stress response of alpha-proteobacteria

本项目将阐明小蛋白在α变形杆菌应激反应中的作用。它将首先关注小DUF1127（未知功能结构域）蛋白，该蛋白存在于许多α和γ变形菌中，并且在结构上与真核RNA结合蛋白的结构域相关。DUF1127结构域包含40个氨基酸（aa），这些氨基酸要么代表一个几乎完整的45-50 aa的蛋白质，要么代表60-75 aa的蛋白质的c端结构域，在极少数情况下高达100 aa。我们将首先重点阐明两个DUF1127蛋白RSP_6037和RSP_0557在兼性光合细菌球形Rhodobacter sphaeroides中的功能，这两个蛋白已经有了初步的数据。RSP_6037与4个sRNAs （CcsR1-4）共转录，在单线态氧和其他应激反应中起作用。在Alphaproteobacteria基因组中，DUF1127蛋白基因通常与CcsR同源基因共定位。在sphaeroides中，sRNAs是由RNase e介导的初级转录本加工产生的，RSP_6037蛋白强烈影响其水平。第二个DUF1127蛋白RSP_0577也受到多种胁迫的诱导，在胁迫防御中起作用，对CcsR sRNAs的水平有中等影响。虽然RSP_6037是必需的，但RSP_0557不是必需的，也不是操作子的一部分。我们想验证DUF1127蛋白是RNA结合蛋白的假设，它可能影响RNA加工。然而，我们也将考虑DUF1127蛋白与DNA或其他蛋白的相互作用。我们将量化不同生长条件下DUF1127蛋白的数量，分析其对全局RNA水平的影响，并确定生物学功能所需的最小肽序列（与Z1和Z2项目合作）。我们将与Z2合作进行rna -蛋白结合研究，或者，如果证实了与DNA的相互作用，则进行DNA-蛋白结合研究，并绘制转录组或基因组上的结合位点。在初步数据的基础上，DUF1127蛋白的确切功能机制有待进一步阐明。我们也想解决DUF1127蛋白的结构，也与配体结合，与Harald Schwalbe合作。稍后，我们还将讨论DUF 1127蛋白在其他α -变形菌中的功能，特别是那些被优先项目的其他成员研究过的蛋白。DUF1127蛋白在不同的物种中是否具有非常相似的功能，或者它们是否具有不同的功能，这将是一件有趣的事情。由于很可能还有其他小蛋白在应激反应中发挥重要作用，因此我们想要鉴定单线态氧和热应激下球形圆叶藻（R. sphaeroides, Z1）的肽穹。进一步的小蛋白将被选择用于功能分析，优先考虑在其他物种中检测到的同源蛋白。

This project will elucidate the role of small proteins in the stress response of Alphaproteobacteria. It will initially focus on the small DUF1127 (domain of unknown function) proteins, which are found in many Alpha- and Gammaproteobacteria and are structurally related to domains from eukaryotic RNA binding proteins.The DUF1127 domain comprises 40 amino acids (aa), which either represent almost a complete protein of 45-50 aa or the C-terminal domain of proteins with 60-75 aa, in rare cases up to 100 aa. We will first concentrate on the elucidation of the function of the two DUF1127 proteins RSP_6037 and RSP_0557 in the facultative photosynthetic bacterium Rhodobacter sphaeroides, for which preliminary data are available. RSP_6037 is co-transcribed with 4 sRNAs (CcsR1-4) with a function in singlet oxygen and other stress responses. DUF1127 protein genes often co-localize with genes for CcsR homologs in genomes from Alphaproteobacteria. In R. sphaeroides the sRNAs are generated by RNase E-mediated processing from the primary transcript and the RSP_6037 protein strongly affects their level. The second DUF1127 protein, RSP_0577, is also induced by several stresses, plays a role in stress defense and has a moderate effect on the levels of the CcsR sRNAs. While RSP_6037 is essential, RSP_0557 is not essential and not part of an operon. We want to test the hypothesis that the DUF1127 proteins are RNA binding proteins, which may affect RNA processing. However we will also consider an interaction of DUF1127 proteins to DNA or to other proteins.We will quantify the amount of DUF1127 proteins under different growth conditions, analyze their effect on global RNA levels and define the minimal peptide sequence required for the biological function (collaboration with the Z1 and Z2 projects). We will perform RNA-protein binding studies or, if rather interaction to DNA is confirmed, DNA-protein binding studies and map the binding sites on the transcriptome or genome in collaboration with Z2. Based on the preliminary data the exact mechanisms of function of DUF1127 proteins should be elucidated. We also like to solve the structure of the DUF1127 proteins, also in combination with the ligand, in collaboration with Harald Schwalbe. Later on we will also address the function of DUF 1127 proteins in other Alpha-proteobacteria, preferentially those studied by other members of the priority program. It will be interesting to find out, whether DUF1127 proteins serve a very similar function in different species or whether they can adopt different functions.Since it is highly likely that also other small proteins have important functions in stress responses, we want to identify the peptidome of R. sphaeroides (Z1) in response to singlet oxygen and heat stress. Further small proteins will be selected for functional analysis, preferentially those with homologs detected in other species in course of the priority program.