Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

Abstract

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.

Ribosome RNA Assembly Intermediates Visualized in Living Cells

“…35 Third, these helices are similar to those that are either improperly folded or interact with protein partners that are missing in immature 30S subunits created by deleting the ribosome late-assembly factors RbfA and RimM. 4,36 Finally, six of the eight proteins that bind directly to the unfolded helices were defined as late-binding tertiary proteins in the original Nomura maps 31 and have recently been shown to bind during late stages of ribosome assembly in vivo . 3 (Fig.…”

Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM

“…16S RNA bridges B5 and B6 have intersubunit contacts on the 30S subunit contributed entirely by helix 44. Helix 44 has been shown to be organized late during the assembly of the 30S subunit and has been found to be disordered (18,19) or distorted (20)(21)(22) in structures of some of the assembly intermediates of the small subunit from various species.…”

The C-terminal helix in the YjeQ zinc-finger domain catalyzes the release of RbfA during 30S ribosome subunit assembly

“…In particular, the folding events involving the maturation of the decoding center in the 30S subunit and PTC in the 50S subunit, occurring at the late stages of assembly (Jomaa et al 2011a(Jomaa et al , 2014Guo et al 2013;Leong et al 2013;Li et al 2013) have a strong tendency to fall into local energy minima. Therefore, bacterial cells have acquired a number of protein factors that are dedicated to assist the folding of these motifs critical for ribosome function (Wilson and Nierhaus 2007); however, it is still not understood how they perform their function.…”