Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis

Abstract

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

FIGURE 1.

KsgA binds to the platform of the 30S ribosomal subunit. A, cryo-EM reconstruction of the translationally inactive 30Si ribosomal subunit at 15.5 Å resolution. The crystal structure of the 30S subunit from E. coli was fitted into the density with head and body as rigid bodies (blue ribbon). Helix 44 of the 16 S rRNA is colored magenta. B, reconstruction of the KsgA-30Si complex at 13.5 Å resolution. The density for the KsgA is colored red. C, structure of the reactivated 30S ribosomal subunit. The structure shows density for the full helix 44 of the 16 S rRNA (magenta).

FIGURE 2.

Contact sites of KsgA on the 30Si subunit platform. A, crystal structure of E. coli KsgA (Protein Data Bank code 1QYR, N-terminal domain, red; C-terminal domain, dark red) is fitted into the cryo-EM density (gray mesh). The methyl donor S-adenosyl-l-methionine is shown in yellow (modeled from Ref. 26). KsgA is bound to helix 24 (orange), 27 (blue), and 45 (cyan) of the 16 S rRNA (Protein Data Bank code 2AVY). The substrate adenosine bases methylated by KsgA are shown in magenta (arrow). B, rotated view of A. The crystal structure KsgA is shown in surface representation colored by surface charge (calculated with DelPhi). A conserved positively charged surface patch interacts with the 16 S rRNA (helix 45, cyan; helix 24, orange). C, rotated view of A (without the cryo-EM density). Positively charged residues important for 30S binding are shown as spheres (²²¹RRK²²³, blue; Arg-248, purple). D, superposition of the core catalytic domain of DNA methyltransferase M.TaqI (Protein Data Bank code 1G38, blue) in complex with substrate DNA (gray) onto KsgA (red, same view as in C). In the M.TaqI structure, the substrate adenosine base (green) is flipped out into the catalytic center.

FIGURE 3.

KsgA sterically excludes helix 44 from its position in the mature 30S subunit. KsgA (red) bound to the 30S subunit (blue, helix 28 yellow) at helix 45 (cyan) overlaps with helix 44 (orange) as observed in the crystal structure of the 30S subunit used for fitting the cryo-EM density (Protein Data Bank code 2AVY). Inset, overview of the KsgA-30Si structure (30S, yellow; KsgA, red) with the head removed (gray surface capping) in the same orientation.

FIGURE 4.

Binding sites of Era and KsgA on the 3′ end of the rRNA. KsgA (red) binds at helix 45 (cyan, A1518 and 1519 magenta) of the 30S subunit (blue). Era (Protein Data Bank code 1X18, green) is bound between the platform and the head contacting helix 28 (yellow) (51). Helix 44 is shown in orange. (The 3′ end of the 16 S rRNA is labeled with an asterisk.)

FIGURE 5.

Schematic illustration of the conformational rearrangements of the 30S subunit rRNA controlled by KsgA. The 30S subunit is shown colored by domain (5′ domain, 30 body, yellow; central domain, 30S platform, green; 3′ domain, 30S head, blue). The 3′ minor domain rRNA helix 44 and helix 45 undergoing conformational changes are shown schematically in blue. KsgA is shown in red. The methylated bases in helix 45 are indicated in magenta. The binding sites of RbfA, RsgA, and Era are indicated by blue and orange circles, respectively. The arrow indicates the cleavage of the 17 S rRNA following dissociation of KsgA. (for explanation see text).