Conformational switch in the ribosomal protein S1 guides unfolding of structured RNAs for translation initiation

Figures

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  Figure 1.. RNA-binding and –chaperone activity of rS1 and its truncated versions. (A) Schematic overview of the rS1 constructs used in this study. Fragments are aligned according to their amino acid sequence. Sequence identities compared to domain D3 are displayed on the top. (B) Electrophoretic mobility shift assays (EMSAs) of selected rS1 constructs, using full-length add Asw (4 μM) as RNA substrate. Increasing protein concentrations are depicted on the top of the gels. The EMSAs were performed on discontinuous gels and only the resolving phases are displayed. Bands reporting on complex formation or free RNA are labeled with RNP or Asw, respectively. Two bands are observed for the free RNA, as the ligand-free riboswitch adopts two conformations (apoA and apoB) (38). In case of the full-length rS1 these states are also resolved for the RNP, since the EMSA was performed at higher powers in order to ensure migration of the complex into the resolving phase of the gel. In addition for the full-length rS1 a high-molecular species was observed (<5%) that did not migrate into the resolving phase of the discontinuous gel (not displayed). (C) Schematic overview of add Asw constructs. Asw-42 is…

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  Figure 2.. Comparison of the mRNA-binding core domains by NMR and sequence. (A) 1H–15N-BEST-TROSY spectra of rS1 constructs as indicated. The spectrum of rS1-D345 was acquired at 35°C and 950 MHz. All other spectra were acquired at 30°C and 600 MHz. (B) Domain sequences were aligned using the Multiple Sequence Alignment tool from Clustal Omega (68–70). Sequence identities in comparison to domain D3 are displayed on the right. Secondary structure elements were computed using SWISS-MODEL (71–74) and are displayed on the top. Identical residues are highlighted.

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  Figure 3.. Interaction of rS1-D34 with Asw-14. (A) Superposition of 1H-15N-BEST-TROSY acquired at 35°C and 600 MHz. NMR titration was performed with 100 μM 15N-labeled rS1-D34. Unlabeled Asw-14 was added stepwise with ratios ranging from 0 to 5.5 equivalents. The molar ratio ([RNA]:[protein]) is color coded within the superimposed spectra. (B) Cartoon representations of D3 and D4 model structures that were generated in SWISS-MODEL (71–74) using the PDB entry 2KHI (18) as template for homology modeling. Observed chemical shift perturbations of binding site reporters are plotted and color coded according to CSP values. The surface is displayed and solvent exposed residues are additionally shown as sticks. Surface binding site reporters are annotated and basic and aromatic residues are highlighted. (C) Chemical shift perturbations, calculated as described in the method section, between free and bound rS1-D34 are plotted as function of rS1-D34 sequence. Horizontal line indicates threshold value that was used to identify binding site reporters (53). Asterisks mark residues that are undetectable due to RNA-induced exchange broadening. Missing values represent either prolines or undetectable…

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  Figure 4.. The two conformational states of rS1-D5. (A) 1H–15N-HSQC spectra were acquired at the displayed temperatures and at 900 MHz. (B) Annotated resonance assignment for D5res-α. The spectrum (acquired at 12°C and 900 MHz) is identical to the spectrum in panel (A) but to exclusively display the predominantly unstructured D5 state, the 1H–15N-HSQC is plotted at higher contour levels.

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  Figure 5.. Structural characteristics of the D5res-α state. The chemical shifts of backbone atoms are sensitive for local structure. Their deviations from random coil values can be used for determination of secondary structure based on experimental data. In the upper plot the fractional secondary structure as calculated from TALOS-N is plotted against the protein sequence (59), where an α-helix is found between A424-I429. The lower plots display 1H, 15N and 13C secondary chemical shifts as a function of residue number. They were calculated as Δ = δobs − δrc (58), where δobs are the observed chemical shifts and δrc are the random coil chemical shifts. δrc were generated using the Javascript provided by Alex Maltsev, on the website of the University of Copenhagen (http://www1.bio.ku.dk/english/research/bms/research/sbinlab/groups/mak/randomcoil/script; 18 April 2018). The expected secondary structure element is indicated within the plot with arrows. All secondary chemical shift values indicate α-helical structure of the sequence stretch A421-G432. The largest values coincide with the AERERI sequence, strongly pointing towards α-helical structure of this particular sequence. In case of ΔCB and…

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  Figure 6.. Structural heterogeneity of D5 in presence of preceding domains and RNA. (A) The 1H–15N-HSQC of rS1-D5 was acquired at 35°C and 950 MHz and an excerpt of tryptophan indole region is displayed to illustrate the res-α state. (B) and (C) displays excerpts of tryptophan indole region of rS1-D345 in the absence and presence of two equivalents Asw-42, respectively. For the NMR titration experiments, 1H–15N-BEST-TROSY (NS = 96) spectra were acquired at 35°C and 950 MHz. Unlabeled Asw-42 was added stepwise with ratios ranging from 0 to 2 equivalents. In the presence of RNA a huge intensity decrease of rS1-D345 resonances was observed. Hence, the titration end point was additionally acquired with more number of scans (NS = 256). This spectrum was used for determination of D5-populations and is displayed in panel (C). Positive 1D projections are displayed within the spectra. W-Hϵ1 resonances of D5res-αare annotated in red and of D5OB in blue. (D) Schematic overview of tryptophan-mediated domain stabilization. (E) The populations of the two D5 states are plotted against rS1 constructs. Populations of each reporter signal were determined from their intensities as…

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  Figure 7.. Intrinsic structural heterogeneity of domain D5. Schematic representation of the D5OB and D5res-α conformational states within the rS1 constructs (A) rS1-D5, (B) rS1-D45 and (C) rS1-D345. The populations are displayed in the scheme. (D) D5 populations in the absence and presence of Asw-42.