Ribosome assembly is a remarkably complex cellular process. In the yeast large subunit (60S) alone, it requires the ordered association and folding of 3 rRNAs and 46 r-proteins as the nascent ribosomes move from the nucleolus, to the nucleus, and finally to the cytoplasm. Like workers building a house, there are over 200 proteins, knows as assembly factors (AFs), that coordinate the construction of these gigantic cellular machines. Each AF interacts with a different part of the pre-ribosome at points along the construction timeline, creating a tightly controlled manufacturing line. There has been much prior work to elucidate the hierarchy and timeline of involved AFs to show how the pre-ribosomal mass bends and grows into a mature, functional subunit. Adding to this, Sanghai, Miller, et al. have recently used cryo-EM to visualize one of the earliest states of yeast 60S ribosomal subunit assembly in the nucleolus, termed 27SB.
After starving yeast to stall ribosome assembly, they carefully affinity purified tagged AFs known to interact with the 27SB state. Cryo-EM of these complexes allows placement of 21 different AFs onto the maturing rRNAs. Importantly, many of these AFs appear to be shielding solvent exposed rRNA elements from forming premature contacts with other parts of the maturing ribosome, implying a direct mechanism of action for these AFs. Additionally, the authors note a surprising similarity between the binding of an early AF, Erb1, and a later factor, Nop53, occupying a similar location. This ‘mimicry’ allows Erb1 to strongly occlude Nop53 until it is forcibly dissociated at later assembly points, suggesting a mechanism for temporal control of AF interaction. Adding to the complexity of it all, multiple sub-states of 27SB were solved, illuminating distinct pathways toward the formation of the peptide exit tunnel. It was already known that there are several correct ‘paths’ a ribosome can take to adulthood, so it is not surprising the authors identified these states even after their specific enrichment.
More broadly, this paper demonstrates the power of cryo-EM to solve the structure of intricate macromolecular complexes, delivering a picture that suggests many new mechanisms and interactions. Furthermore, cryo-EM and image classification can reveal heterogeneity in ‘pure’ samples, giving evidence for alternate pathways and conformations. Lastly, the biochemical effort required to purify these small populations of complexes out of the cellular milieu is always particularly impressive!