A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate
- Department of Biological Sciences, Columbia University, United States
- Department of Biochemistry and Molecular Biophysics, Columbia University, United States
- Department of Cell Biology, SUNY Downstate Medical Center, United States
- Structural Biology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Spain
Figures
Figure 1 with 2 supplements
Dicistroviridae genome organization, in vitro complex formation and cryo-EM maps.
(A) Top, schematic representation of the genome organization of Dicistroviruses. The approximate genomic lengths of the different components are indicated by the arrows. Bottom, detailed view of the …
Figure 1—figure supplement 1
Cryo-EM representative images and classification workflow.
(A) Two examples of aligned micrographs used for image processing. Data collection in thick ice was instrumental to avoid complex disassembly and preferential orientation. (B) Representative …
Figure 1—figure supplement 2
FSC correlation curves, local resolution and model validation.
Top, class-1. (A) Gold-standard Fourier Shell Correlation (FSC) curves between half-maps independently refined in Relion3. Global resolution by the 0.143 cutoff criterium was estimated to be 3.3 Å. …
Figure 2 with 1 supplement
The 5'-UTR-IRES domain I engages ribosomal proteins RACK1 and uS3.
(A) Overview of the 40S–5'-UTR-IRES–eIF3 map, with 40S and eIF3 depicted gray and 5'-UTR-IRES in blue. (B) Detailed view of the cryo-EM density of the 40S–5'-UTR-IRES–eIF3 map centered around …
Figure 2—figure supplement 1
Structurally derived secondary structure diagram for the 5'-UTR-IRES.
Secondary structure diagram for the 5'-UTR-CrPV IRES derived from the cryo-EM structure.
Figure 3
The 5'-UTR-IRES domain II is formed by a dual hairpin that mediates eIF3 recruitment.
(A) Overview of the 40S–5'-UTR-IRES–eIF3 cryo-EM map with 40S colored gray, eIF3 red and 5'-UTR-IRES blue. On the right, a zoomed view centers around 5'-UTR-IRES domain II. (B) Detailed view of the …
Figure 4
Non-canonical base pairing in 5'-UTR-IRES domain III assists on P-site access.
A) Overview of the 40S–5'-UTR-IRES–eIF3 cryo-EM map with 40S and eIF3 colored gray and 5'-UTR-IRES blue. On the right, a detailed view of the E-site, where 5'-UTR-IRES domain III is placed, shows the…
Figure 5
A 40S head swiveling movement ‘locks’ 5'-UTR-IRES on the 40S, inducing a compact eIF3 configuration.
(A) Cryo-EM maps obtained for the two classes present in the 40S–5'-UTR-IRES–eIF3 dataset with 40S colored gold, eIF3 red and 5'-UTR-IRES blue. The positions of the latch, eIF3d and the swiveling …
Figure 6 with 1 supplement
The 5'-UTR-IRES requires TC, eIF1 or eIF1A to assemble a functional initiation complex via an uAUG intermediate.
(A) Toe-print analysis of 48S initiation complexes assembled on 5'-UTR-IRES in an in vitro reconstituted system. eIF2 delivers Met-tRNAiMet to the uAUG (lane 2) and requires the presence of eIF1 or e…
Figure 6—figure supplement 1
Control toe-print experiments.
(A) Toe-print analysis for 5'-UTR-IRES, 5'-UTR-IRES–eIF3 and 40S/5'-UTR-IRES–eIF3 complexes (lanes 1, 2 and 3, respectively). No toe-print signal was detected that is ascribable to uAUG or annotated …
