Co-Transcriptional Ribosome Assembly in Real-Time

Abstract

The bacterial ribosome is a large macromolecular machine composed of three large RNA molecules and more than 50 proteins, which are co-transcriptionally assembled in a living cell within 1-2 minutes.The molecular mechanism of assembly has extensively been studied with pre-transcribed ribosomal RNA (rRNA) mainly using ensemble methods, in which binding of each ribosomal protein (r-protein) is detected as the average state of all populations in which it is present. Because active transcription at the correct speed is essential for ribosome assembly at physiological conditions, the molecular mechanism of ribosome assembly has to be understood co-transcriptionally.Here we present a novel single-molecule fluorescence based approach in which transcription and folding of single rRNA molecules and the binding of r-proteins to them can be visualized simultaneously in real-time.Using zero-mode waveguide instrumentation, we could monitor transcription progression of single RNA molecules of several hundred nucleotides.Studying the co-transcriptional binding of the primary r-protein S15 to the central domain shows that besides short lived binding events (few seconds lifetime), specific S15 binding (>30 seconds lifetime) can occur shortly after transcription of its binding site. In contrast, co-transcriptional binding of primary protein S7 to the entire 3’-domain is much more dynamic with some RNA molecules being unable to stably bind S7 (seconds lifetime) while others can eventually bind S7 specifically, yet can do so only minutes after transcription at 20 degrees Celsius.Overall, using a novel approach to observe in real-time binding of single r-proteins to transcribing r-RNA, we see that co-transcriptional folding of the central 16S rRNA domain is efficient allowing immediate protein binding, while 3’-domain folding is inefficient, indicating that other r-proteins or assembly factors such as RNA helicases must increase folding efficiency in the cell (at least at low temperature).