Figure 7.
Ligatin-mediated 48S complex formation on SV 26S mRNA with noninitiator tRNAs, and interaction of Ligatin and MCT-1/DENR with tRNAMeti on the 40S subunit. (A) Mutant SV 26S mRNAs with AUG50 replaced by CUG or GUG. (B) Analysis of Ligatin's ability to promote 48S complex formation on mutant SV 26S mRNAs (shown in A) in the presence of tRNAMeti and cognate Leu-tRNALeu and Val-tRNAVal, assayed by toe-printing. (C) Analysis of Ligatin's ability to promote 48S complex formation on CAC codons preceding AUG50 of wild-type SV 26S mRNA in the presence of cognate His-tRNAHis, assayed by toe-printing. (B,C) Lanes C, T, A, and G depict corresponding cDNA sequences. Positions of AUG, CUC, and GUG codons, full-length cDNA, and toe prints corresponding to ribosomal complexes are indicated. (D) UV-cross-linking of Ligatin and MCT-1/DENR with tRNAMeti in 48S complexes assembled on ΔII CSFV IRES with 32P-labeled tRNAMeti containing 4-thioU and purified by SDG centrifugation, assayed by SDS-PAGE and autoradiography. The positions of Ligatin, DENR, cross-linked ribosomal protein, and molecular weight markers are indicated.