Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA

Abstract

Reconstitution of translation elongation from purified components confirmed that ribosomes that assembled on the Cricket paralysis virus intercistronic internal ribosomal entry site (IRES) without the involvement of initiation factors or initiator tRNA were active in elongation and are, therefore, true initiation complexes. The first elongation cycle occurred without peptide bond formation on 80S ribosomes that did not contain tRNA in the P site. It required elongation factors 1A and 2 and A site-cognate aminoacylated tRNA. Cycloheximide arrested ribosomes on the IRES only after two cycles of elongation, when the first deacylated tRNA reached the E-site after translocation from the A-site.

Figure 1

Purification of elongation factors. (a) Typical chromatogram of eEF1 on Superdex G200. The positions of eEF1A/1Bαβγ (eEF1H), eEF1A/1Bαγ, and eEF1A are indicated. (b,c) Resolution by SDS-PAGE of eEF2 (b, lane 2), eEF1A/1Bαβγ (c, lane 3), and eEF1A/1Bαγ (c, lane 4). The positions of molecular weight markers are indicated to the left of lane 1. Subunits of eEF1H are indicated to the left of lane 3. SDS-PAGE gels were stained with Coomassie blue.

Figure 2

Elongation factor and tRNA requirements for translocation of ribosomes assembled on the IRES in the presence of the indicated components. The positions of toeprints relative to nucleotides +1 of the CCU triplet present in the P site before translocation are indicated to the right of each panel and schematically below the CrPV ORF2 sequence. Lanes C, T, A, and G depict the CrPV sequence.

Figure 3

Template dependence of cycloheximide-mediated arrest of ribosomal translocation. Toeprints caused by arrested 48S complexes and 80S ribosomes on β-globin (a), (CAA)n-GUS (b), and CSFV (c) IRES-NS' mRNAs in RRL in the presence of 1 mM GMPPNP or 500 μg/mL of cycloheximide, as indicated. The positions of toeprints relative to ntucleotides +1 of the start codon are indicated to the right of each panel. Appropriate cDNA sequences are on the left half of each panel.

Figure 4

tRNA-dependent translocation of ribosomes assembled on the IRES. Toeprint analysis of translocation of ribosomes assembled on CrPV-FFM (a), CrPV-MMF (b), and CrPV-MF (c) mRNAs in the presence of aminoacylated tRNAs and other components as indicated. The positions of toeprints relative to nucleotides +1 of the CCU triplet present in the P site before translocation are indicated to the right of each panel. Lanes C, T, A, and G depict appropriate CrPV mutant sequences.

Figure 5

Template-dependent synthesis of [³⁵S]methionine-containing aminoacyl- or peptidylpuromycin by 80S ribosomes assembled from 40S and 60S subunits on the CrPV IRES in the presence of eEFs 1A and 2 with mRNAs and aminoacylated tRNAs as indicated.