Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

Abstract

YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 39 end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.

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

“…Filtration assays (Supplemental Fig. S3; left panel) performed with buffers containing a concentration of NH 4 Cl ranging from 60 to 600 mM showed that at 300 mM NH 4 Cl, YjeQ bound to the 30S subunit at ∼1:1 ratio, which is the stoichiometry that has been previously established for the 30S+YjeQ complex (Daigle and Brown 2004;Himeno et al 2004;Guo et al 2011;Jomaa et al 2011b). …”

“…Their roles in the maturation of the functional core of the 30S subunit may entail facilitating proper 17S rRNA folding, assisting processing of the rRNA, or mediating protein-RNA interactions. Cryo-electron microscopy (cryo-EM) (Sharma et al 2005;Datta et al 2007;Guo et al 2011;Jomaa et al 2011b) revealed that at least three factors, Era, YjeQ, and RbfA bind at or in close proximity to the decoding center at sites that are not overlapping, indicating that simultaneous binding is stereochemically possible. Consistently, genetic experiments (Bylund et al 1998(Bylund et al , 2001Inoue et al 2003Inoue et al , 2006Campbell and Brown 2008) suggested that these factors operate in conjunction rather than independently.…”

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

“…To globally assay changes in preribosomal protein constituents upon depletion of L7 or L8, we used iTRAQ, a semiquantitative mass spectrometric method ( Fig. 4; Ross et al 2004;Merl et al 2010;Jomaa et al 2011). In the case of depletion of L8, the iTRAQ data were confirmed using another tagged assembly factor, Noc2, to purify preribosomes (Supplemental Fig.…”

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

“…This argues that RbfA has to act before KsgA in the cascade of biogenesis factors interacting with the pre-30S subunit. Furthermore, a recent structure of RsgA, which is the release factor of RbfA, on the 30S subunit shows RsgA in a conformation that would sterically exclude KsgA (54,55). This suggests that RsgA removes RbfA before KsgA can bind to the immature 30S subunit.…”