RbfA Is Involved in Two Important Stages of 30S Subunit Assembly: Formation of the Central Pseudoknot and Docking of Helix 44 to the Decoding Center

Abstract

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.

Mechanism of mitoribosomal small subunit biogenesis and preinitiation

“…2b and Supplementary Video 2 ), which is enabled by a pivot point between the KH-like domain and CTD. The conformational change appears to be a unique feature of RBFA, which was not reported for bacterial counterparts 10 , 25 . METTL15 would clash with RBFA-in from pre-SSU-2 (Extended Data Fig.…”

Differential Proteomic Analysis of Listeria monocytogenes during High-Pressure Processing

“…RbfA is associated with 16S rRNA but not 70S ribosomes or polysomes, and is essential in late-30S ribosomal subunit maturation [ 27 ]. Recently, RbfA and RimM were found to be involved in two distinct 30S assembly stages consisting of the early development of the central pseudoknot with the folding of the head and further docking of helix 44 to form important inter-subunit bridges in the mature structure at a later stage [ 28 ]. A comprehensive review revealed that increased RbfA synthesis could moderately attenuate △ rimM mutation.…”

Increasing Complexity of Ribosomes and Their Biogenesis

“…Maksimova, Korepanov, Kravchenko, Baymukhametov, Myasnikov, Vassilenko, Afonina, and Stolboushkina classified the bacterial 30S ribosomal subunit according to structure in response to depleting the assembly factor RbfA. The results indicate that RpfA stabilizes the 30S pseudoknot (a universal feature of the small ribosomal subunit across the evolutionary spectrum) and assists the docking of 16S rRNA Helix 44 into the decoding center [ 21 ].…”