Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 1998 Jan 1;12(1):67–83. doi: 10.1101/gad.12.1.67

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 1998, Cold Spring Harbor Laboratory Press

PMC Copyright notice

Binding of 40S subunits and assembly of 48S complexes on HCV and CSFV mutant IRESs. (A) Wild-type HCV (nucleotides 1–349)–CAT (lanes 1–3) and mutant HCV (nucleotides 1–349Δ172–227)–CAT (lanes 4–6) were incubated under standard conditions with 40S ribosomal subunits (lanes 2,3 and 5,6) and with eIF2, eIF3, Met–tRNA_i^Met and GMP–PNP (lanes 3,6). (B) Mutant HCV (nucleotides 1–380Δ26–67)–CAT (lanes 1–3), (nucleotides 1–380Δ172–227)–CAT (lanes 4–6) and (nucleotides 1–380Δ229–238) (lanes 7–9) RNAs were incubated with 40S subunits (lanes 2,3,5,6,8,9) and with eIF2, eIF3, Met–tRNA_i^Met, and GMP–PNP (lanes 3,6,9). Reaction conditions are described in Materials and Methods. A primer (5′-GCAACTGACTGAAATGCC-3′) was annealed to the CAT cistron ∼80 nucleotides downstream of the HCV initiation codon and was extended with AMV–RT. Primer extension was arrested at sites indicated on the right either as nucleotides in the HCV IRES or as positions relative to the A of the HCV initiation codon. Reference lanes C, T, A, and G depict the HCV–CAT sequence. The junction between the HCV IRES and the CAT cistron is indicated. This gel was exposed to x-ray film longer than that shown in A to show the differences in the intensity of stops on various mutant RNAs. (C) Model of the CSFV pseudoknot and adjacent nucleotides showing the substitutions present in the mutants UC₃₂₅, GA₃₅₇, and UC₃₂₅GA₃₅₇ and the deletions made in the mutants ΔA₁₄₅–U₁₄₈ and ΔA₃₄₉–A₃₅₃. In this model, helix I of the pseudoknot comprises nucleotides 129–142 and 331–346, and helix II comprises nucleotides 323–329 and 354–360. (D) CSFV (nucleotides 1–442)–NS′ RNAs containing the mutations UC₃₂₅ (lanes 1–3), GA₃₅₇ (lanes 4–6), UC₃₂₅GA₃₅₇ (lanes 7–9), and ΔA₃₄₉–A₃₅₃ (lanes 10–12) were incubated with 40S subunits (lanes 2,3,5,6,8,9,11,12) and with eIF2, eIF3, Met–tRNA_i^Met and GMP–PNP (lanes 3,6,9,12).

Binding of 40S subunits and assembly of 48S complexes on HCV and CSFV mutant IRESs. (A) Wild-type HCV (nucleotides 1–349)–CAT (lanes 1–3) and mutant HCV (nucleotides 1–349Δ172–227)–CAT (lanes 4–6) were incubated under standard conditions with 40S ribosomal subunits (lanes 2,3 and 5,6) and with eIF2, eIF3, Met–tRNA_i^Met and GMP–PNP (lanes 3,6). (B) Mutant HCV (nucleotides 1–380Δ26–67)–CAT (lanes 1–3), (nucleotides 1–380Δ172–227)–CAT (lanes 4–6) and (nucleotides 1–380Δ229–238) (lanes 7–9) RNAs were incubated with 40S subunits (lanes 2,3,5,6,8,9) and with eIF2, eIF3, Met–tRNA_i^Met, and GMP–PNP (lanes 3,6,9). Reaction conditions are described in Materials and Methods. A primer (5′-GCAACTGACTGAAATGCC-3′) was annealed to the CAT cistron ∼80 nucleotides downstream of the HCV initiation codon and was extended with AMV–RT. Primer extension was arrested at sites indicated on the right either as nucleotides in the HCV IRES or as positions relative to the A of the HCV initiation codon. Reference lanes C, T, A, and G depict the HCV–CAT sequence. The junction between the HCV IRES and the CAT cistron is indicated. This gel was exposed to x-ray film longer than that shown in A to show the differences in the intensity of stops on various mutant RNAs. (C) Model of the CSFV pseudoknot and adjacent nucleotides showing the substitutions present in the mutants UC₃₂₅, GA₃₅₇, and UC₃₂₅GA₃₅₇ and the deletions made in the mutants ΔA₁₄₅–U₁₄₈ and ΔA₃₄₉–A₃₅₃. In this model, helix I of the pseudoknot comprises nucleotides 129–142 and 331–346, and helix II comprises nucleotides 323–329 and 354–360. (D) CSFV (nucleotides 1–442)–NS′ RNAs containing the mutations UC₃₂₅ (lanes 1–3), GA₃₅₇ (lanes 4–6), UC₃₂₅GA₃₅₇ (lanes 7–9), and ΔA₃₄₉–A₃₅₃ (lanes 10–12) were incubated with 40S subunits (lanes 2,3,5,6,8,9,11,12) and with eIF2, eIF3, Met–tRNA_i^Met and GMP–PNP (lanes 3,6,9,12).