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.
Highlights
The combination of genetic modification with cryo-electron microscopy (cryo-EM) analysis is widely used to identify the role of protein factors in assisting specific steps of the ribosome assembly process
The approach consists of creating a strain with knockout or depletion of a single assembly factor gene to disable or slow down the ribosome biogenesis process
The main conclusion that was made based on this type of experiment was that most of the assembly factors assist in the maturation of the functional core of the 30S subunit—the decoding center [27]—located at the upper part of the body and the lower part of the head
Summary
Ribosome biogenesis is a complicated multi-stage process comprising synthesis and maturation of ribosomal RNA (rRNA), the synthesis, modification, and folding of ribosomal proteins (r-proteins), and consecutive assembly of ribosomal subunits. Ribosome biogenesis is facilitated by numerous protein assembly factors: GTPases, RNA helicases, molecular chaperones, rRNA modification enzymes, etc. These factors help to avoid kinetic traps during RNA folding [1], assist the attachment of ribosomal proteins and prevent their premature and non-native binding [2]. RbfA has been identified as a bacterial cold shock response protein required for the efficient processing of the 16S rRNA 50 end during the assembly of the
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