Figure 4.

Cis-Acting Sequences Are Crucial to aTI.

(A) IVTT analysis of a novel aTI site derived by insertion of CTG with varying lengths of surrounding sequence (capitalized in bold) to the 5′ UTR of the cyclophilin-40 gene (At2g15790). Each construction also includes 444 nucleotides of cyclophilin-40 sequence 3′ to the ATG. The construction designated ATG contains no 5′ UTR sequence. The differences in size of the aTI-derived products correspond to the differences in insertion sequence length 3′ to CTG, confirming initiation at the introduced aTI site.

(B) EMSA of RNA-protein binding at the Polγ2 CTG aTI site. Bound RNA probe is shown as bands, while free RNA probe migrates to the bottom of the gel (data not shown). RNA probe sequences corresponding to the individual EMSA assays are shown, with protein binding assays conducted with wheat germ extracts. While some low level binding is detected for the antisense RNA, this may be due to similar sequence features present in the antisense comprised of three purines (GAA) followed by CTT and another purine (A). The bottom panel shows results of binding competition experiments with varying proportions of α³²P-labeled/unlabeled RNA probe 2.

(C) Identical EMSA experiment to that shown in (B) with radiolabeled RNA probes 1 to 5, but substituting purified eIF4A-1 overexpression product in place of wheat germ extracts in the RNA binding assay.