Near-physiological in vitro reconstitution of 50S subunit reveals parallel assembly pathway.

Abstract

The ribosome is one of the largest and the most complicated enzymes in cells, and is responsible for protein synthesis in all living organisms on earth. During the bacterial ribosome biogenesis, the ∼5-kb transcriptional products are cleaved into three component ribosomal RNAs, undergo post-transcriptional chemical modifications by a set of enzymes, and fold into ∼150 secondary helices structures, facilitated by the chaperoning and controlling of more than 100 ribosome biogenesis co-factors. Despite its complexity, the bacterial ribosome assembly process takes less than 2 minutes in cells, so the intermediates during the step-wised ribosome assembly process are incredibly short-lived, making it difficult to directly separate and investigate the incomplete ribosomes under native physiological environments. In this work, we report a time-course structure library of ribosome large subunit intermediates accumulated from a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate structures covering the whole assembly process were resolved by cryo-EM single particle analysis using heterogeneous subclassification. Segment behavior analysis reveals that the ribosome intermediates assemble based on fourteen cooperative assembly blocks, including a smallest assembly core reported up to now, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative assembly blocks are found to fold onto the assembly core following a defined set of dependencies. Some of these dependencies are parallel to each other, highlighting the parallel assembly pathways at both early and late assembly stages of the 50S subunit.