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

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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.