Transcription Increases the Cooperativity of Ribonucleoprotein Assembly

Highlights

• Single-molecule assay monitors transcription and RNP assembly in real time

• Proteins sample the pre-rRNA as soon as the binding site is synthesized

• Multiple ribosomal proteins commit the rRNA to assembly during transcription

• All-or-none co-transcriptional RNP assembly mechanism avoids assembly pitfalls

Summary

The synthesis of new ribosomes begins during transcription of the rRNA and is widely assumed to follow an orderly 5′ to 3′ gradient. To visualize co-transcriptional assembly of ribosomal protein-RNA complexes in real time, we developed a single-molecule platform that simultaneously monitors transcription and protein association with the elongating transcript. Unexpectedly, the early assembly protein uS4 binds newly made pre-16S rRNA only transiently, likely due to non-native folding of the rRNA during transcription. Stable uS4 binding became more probable only in the presence of additional ribosomal proteins that bind upstream and downstream of protein uS4 by allowing productive assembly intermediates to form earlier. We propose that dynamic sampling of elongating RNA by multiple proteins overcomes heterogeneous RNA folding, preventing assembly bottlenecks and initiating assembly within the transcription time window. This may be a common feature of transcription-coupled RNP assembly.