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

这个项目将阐明小蛋白在甲型变形杆菌应激反应中的作用。它首先将重点放在小的DUF1127(未知功能结构域)蛋白上，它存在于许多阿尔法和伽马蛋白细菌中，在结构上与真核RNA结合蛋白的结构域相关。DUF1127结构域由40个氨基酸(AA)组成，它们要么代表一个几乎完整的45-50个氨基酸的蛋白质，要么代表60-75个氨基酸的蛋白质的C-末端结构域，在少数情况下长达100个氨基酸。我们将首先集中阐明两个DUF1127蛋白RSP_6037和RSP_0557在兼性光合细菌球形红假单胞菌中的功能，这是有初步数据的。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。DUF1127蛋白基因通常与甲型变形杆菌基因组中CCSR同源基因共定位。在球状乳杆菌中，sRNA是由RNase E介导的初级转录本加工而成的，RSP_6037蛋白强烈地影响着它们的水平。第二个DUF1127蛋白RSP_0577也受多种胁迫诱导，在胁迫防御中发挥作用，对CCSR sRNAs水平有一定的调节作用。虽然RSP_6037是必需的，但RSP_0557不是必需的，也不是操纵子的一部分。我们想检验DUF1127蛋白是RNA结合蛋白的假设，这可能会影响RNA的加工。然而，我们也将考虑DUF1127蛋白质与DNA或其他蛋白质的相互作用。我们将量化不同生长条件下DUF1127蛋白质的数量，分析它们对全球RNA水平的影响，并定义生物功能所需的最小肽序列(与Z1和Z2项目合作)。我们将进行RNA-蛋白质结合研究，或者，如果证实与DNA相互作用，则进行DNA-蛋白质结合研究，并与Z2合作绘制转录组或基因组上的结合位置图。基于初步的数据，DUF1127蛋白的确切作用机制有待阐明。我们还希望与Harald Schwalbe合作，解决DUF1127蛋白质的结构，也与配体结合。稍后，我们还将讨论DUF 1127蛋白在其他阿尔法蛋白细菌中的功能，优先考虑优先计划其他成员所研究的那些蛋白。DUF1127蛋白在不同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，这将是一个有趣的发现。由于很可能还有其他小蛋白在应激反应中具有重要功能，我们想要鉴定球形螺旋藻Z1对单线态氧和热胁迫的反应。将选择更多的小蛋白进行功能分析，优先选择那些在优先计划过程中在其他物种中检测到同源物的蛋白。

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.