Ruprecht-Karls-Universit├Ąt Heidelberg
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Bukau_Kramer0118 - Scientist (f/m) / PhD position
Project no:

Project leader:

Project supervisor:
Bukau, Bernd
Application deadline:
15. Jan 2019
Start of PhD project:
1. Mar 2019

Project description:

Network analysis of co-translationally acting factors mediating membrane targeting of proteins in E. coli
According to established knowledge, E. coli employs two main pathways to translocate newly synthesized proteins into and across the membrane: Insertion of inner membrane proteins initiates co-translationally by ribosome binding of the signal recognition particle (SRP). Translocation across the membrane is independent of ribosome docking, driven by translocon-docked SecA and for some proteins facilitated by the anti-folding chaperone SecB.
Recent evidence from us and other labs suggest that key features of the membrane targeting process in E. coli remain unknown and imply that the clear separation into co- and post-translational translocation modes may be incorrect. First, we recently identified new, unexpected features of SRP mediated translocation, including the skipping of N-terminal targeting signals and the multiple binding of SRP to translating ribosomes1 and second, we discovered that SecA and SecB also engage ribosomes synthesizing membrane proteins2,3.
The proposed project aims at providing a comprehensive understanding of nascent protein selection by co-translationally acting factors that triage proteins to the co- versus post-translational translocation pathways. The study will extend our understanding of protein sorting at the ribosome and reveal how a network employs functional antagonism and redundancy of the factors involved to maximize the efficiency of substrate selection.
1. Schibich, D., Gloge, F., Pohner, I., Bjorkholm, P., Wade, R.C., von Heijne, G., Bukau, B.*, and Kramer, G.* (2016). Global profiling of SRP interaction with nascent polypeptides. Nature 536, 219-223.

2. Huber, D., Rajagopalan, N., Preissler, S., Rocco, M.A., Merz, F., Kramer, G., and Bukau, B. (2011). SecA Interacts with Ribosomes in Order to Facilitate Posttranslational Translocation in Bacteria. Molecular Cell 41, 343-353.

3. Huber, D., Jamshad, M., Hanmer, R., Schibich, D., Doring, K., Marcomini, I., Kramer, G., and Bukau, B. (2017). SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo. Journal of Bacteriology 199.

4. Becker, A.H., Oh, E., Weissman, J.S., Kramer, G.*, and Bukau, B.* (2013). Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes. Nature protocols 8, 2212-2239.

5. Oh, E., Becker, A.H., Sandikci, A., Huber, D., Chaba, R., Gloge, F., Nichols, R.J., Typas, A., Gross, C.A., Kramer, G., et al. (2011). Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147, 1295-1308.

Methods that will be used:
We plan to use Selective Ribosome Profiling (SeRP)4,5, protein biochemistry, cell and molecular biology and bioinformatics tools to (i) explore the nascent chain interactomes of SecA and SecB, (ii) study when during synthesis each factor engages nascent substrates and which molecular features in nascent chains mediate binding (e.g. sequence motifs, hydrophobic stretches, folds and chain lengths) and (iii) to quantitatively compare interaction profiles of nascent chain-engaging factors to characterize the orchestration of factors within the functional network.
Cooperation partners:
Simon Anders (ZMBH, Heidelberg), Sander Tans (Amolf Institute, Amsterdam), Petra van Damme (VIB-UGent Center for Medical Biotechnology, Gent), Hans-Georg Koch (Universit├Ąt Freiburg)
Personal qualifications:
We are seeking for a highly motivated, team-oriented person interested in studying fundamental principles of protein synthesis and transport that has general knowledge of molecular biology and biochemistry. Basic knowledge in bioinformatics is advantageous but not strictly required.
Protein-synthesis and -transport, ribosomes, targeting factors, chaperones, selective ribosome profiling