Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1

Ribosomal protein S1, consisting of six contiguous OB-folds, is the largest ribosomal protein and is essential for translation initiation in Escherichia coli. S1 is also one of the three essential host-derived subunits of Qβ replicase, together with EF-Tu and EF-Ts, for Qβ RNA replication in E. coli. We analyzed the crystal structure of Qβ replicase, consisting of the virus-encoded RNA-dependent RNA polymerase (β-subunit), EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qβ RNA replication. Structural and biochemical studies revealed that the two N-terminal OB-folds of S1 anchor S1 onto the β-subunit, and the third OB-fold is mobile and protrudes beyond the surface of the β-subunit. The third OB-fold mainly interacts with a specific RNA fragment derived from the internal region of Qβ RNA, and its RNA-binding ability is required for replication initiation of Qβ RNA. Thus, the third mobile OB-fold of S1, which is spatially anchored near the surface of the β-subunit, primarily recruits the Qβ RNA toward the β-subunit, leading to the specific and efficient replication initiation of Qβ RNA, and S1 functions as a replication initiation factor, beyond its established function in protein synthesis.


Figure S1
Supplementary Figure S1: Western-blotting of preparations of S1 and its variants used in this work.

Figure S2
Supplemental Figure S2: Amino acid alignment of the OB-folds of ribosomal protein S1 and related proteins. S1OB1~OB6 are the amino acid sequences of the six OB-folds from E. coli ribosomal protein S1.
The electron density corresponding to R3 was not visible. The β-subunit and EF-Tu are colored yellow and red, respectively, and depicted by surface models.  There is sufficient space beyond the surface of the β-subunit, where the R3 of R1-3 of S1 is expected to be located.   Superimposition of the structures of the core Qβ replicase (3; colored cyan) and Qβ replicase containing the N-terminal half of S1 (colored yellow, with the N-terminal half of S1 colored green).

Figure S9
Supplementary Figure S9: Model of the interaction between S1 and the 30S ribosome.
Hydrophobic residues in S2 are colored red in the surface model (right). (B) The N-terminal domains of S1 (R1-2) were docked on the 30S ribosome. The N-terminal domain of S1 is colored green and S2 is colored blue.

SUPPLEMENTARY TEXT
Model of S1 docking onto the 30S ribosome The N-terminal region of ribosomal protein S1 interacts with ribosomal protein S2 in the 30S ribosome (4,5). We searched for the S1-docking surface on S2, using the ZDOCK server (6). For the receptor and ligand, the structures of S2 in the 30S ribosome (7; PDB ID: 2AVY) and of S1 (R1-2) in Qβ replicase ( Figure 3B) were used, respectively. Among the top ten calculated solutions, four have similar binding manners, in which R1 of S1 binds to S2. The structure with the highest rank is shown in Supplementary Figure S5A. The N-terminal helix, α1, is placed on the hydrophobic patch of S2, as seen in the Qβ replicase complex ( Figure 4A, B). This binding manner is also consistent with the results obtained from cross-linking between S1 and S2 in ribosomes (8). We superimposed the model complex of R1-2 of S1 and S2 onto the 30S ribosome (8). In the superimposition, the OB1 fold of S1 is placed next to the 30S ribosomal RNA, and the surface of OB1 of S1 snugly fits on the surface of the 30S ribosome. On the other hand, in the simple superimposition, R2 of S1 clashes with the ribosomal RNA. R2 of S1 would be placed at a different location on the 30S ribosome and/or in a different orientation, using the flexible linker between OB1 and OB2 (α2'-α2) ( Figure 3B). In the model presented here, R2 was manually docked onto the 30S ribosome, considering the results obtained from cross-linking between amino acid residues in R2 and ribosomal proteins in the 30S ribosome (Supplementary Figure S5B).