09 Jan 2018
14:00 - 15:30
Biozentrum/Pharmazentrum, BZ 411, Klingelbergstrasse 50-70, Basel
Early co-transcriptional ribosome assembly in real-time
Vortrag von Dr. Oliver Duss, The Scripps Research Institute, CA, Stanford University, CA
The ribosome is a large macromolecular machine that synthesizes the proteins present in the cell. In bacteria, the ribosome consists of three large RNA molecules and more than 50 proteins, which are co-transcriptionally assembled in a living cell within ~2 minutes. However, it is poorly understood how transcription and RNA folding are coupled to protein binding at the molecular level. Here, we present a novel single-molecule approach that allows simultaneously monitoring transcription elongation and the subsequent binding of fluorescently labeled proteins to the nascent rRNA in real-time. Studying the binding of primary ribosomal protein (r-protein) S7 to the transcribing 3’- domain of the 16S rRNA in real-time, we see how single RNA molecules directly emerging from the RNA polymerase fold and nucleate assembly. The majority of nascent 3’-domain RNA molecules are incompetent to stably bind S7 or can do so only several minutes after transcription and RNA structures remote from the S7 binding site are responsible for RNA misfolding. Higher temperature increases RNA folding efficiency but we do not detect a correlation with the transcription rate. Analyzing the subset of nascent RNA molecules that can stably bind S7, we find that remote RNA structures stabilize the S7 binding site. While it is well established that primary binding proteins, such as S7, are required for the stable binding of secondary ones, we demonstrate that the binding of secondary r-protein S13 reduces the binding dynamics of primary r-protein S7 stabilizing it and remarkably, only by transient binding of S13. Directly correlating transcription, rRNA folding and r-protein binding to single nascent rRNA molecules demonstrates that for certain RNAs co-transcriptional RNA folding can be difficult at low temperature and it will be interesting to investigate the individual contributions of specific ribosome assembly factors to RNA folding efficiency. Furthermore, our results establish that during the course of ribosome assembly, highly dynamic protein-RNA interactions gradually become stabilized by the binding of subsequent r-proteins, in order to eventually obtain a stable ribosome.
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