RESUMO
Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Homeostase , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Sequência de Bases , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Conformação de Ácido Nucleico , Ligação Proteica , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/genética , Subunidades Ribossômicas Menores/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestruturaRESUMO
Our understanding regarding the function of YjeQ (also called RsgA), RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in null strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that these immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particles accumulate in cells. We measured by microscale thermophoresis that YjeQ and Era binds to the mature 30S subunit with high affinity. However, the binding affinity of these factors to the immature particles and of RimM and RbfA to mature or immature particles was weak, suggesting that binding is not occurring at physiological concentrations. These results suggest that in the absence of these factors, the immature particles evolve into a thermodynamically stable intermediate that exhibits low affinity for the assembly factors. These results imply that the true substrates of YjeQ, RbfA, RimM and Era are immature particles that precede the ribosomal particles accumulating in the knockouts strains.
Assuntos
Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Modelos Biológicos , Complexos Multiproteicos , Mutação , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
YjeQ (also called RsgA) and RbfA proteins in Escherichia coli bind to immature 30S ribosome subunits at late stages of assembly to assist folding of the decoding center. A key step for the subunit to enter the pool of actively translating ribosomes is the release of these factors. YjeQ promotes dissociation of RbfA during the final stages of maturation; however, the mechanism implementing this functional interplay has not been elucidated. YjeQ features an amino-terminal oligonucleotide/oligosaccharide binding domain, a central GTPase module and a carboxy-terminal zinc-finger domain. We found that the zinc-finger domain is comprised of two functional motifs: the region coordinating the zinc ion and a carboxy-terminal α-helix. The first motif is essential for the anchoring of YjeQ to the 30S subunit and the carboxy-terminal α-helix facilitates the removal of RbfA once the 30S subunit reaches the mature state. Furthermore, the ability of the mature 30S subunit to stimulate YjeQ GTPase activity also depends on the carboxy-terminal α-helix. Our data are consistent with a model in which YjeQ uses this carboxy-terminal α-helix as a sensor to gauge the conformation of helix 44, an essential motif of the decoding center. According to this model, the mature conformation of helix 44 is sensed by the carboxy-terminal α-helix, which in turn stimulates the YjeQ GTPase activity. Hydrolysis of GTP is believed to assist the release of YjeQ from the mature 30S subunit through a still uncharacterized mechanism. These results identify the structural determinants in YjeQ that implement the functional interplay with RbfA.
