FIG. 1.

Design of LUC reporter mRNAs investigating the translational regulation of TYMV RNA. (A) Diagram of TYMV genomic RNA and subgenomic RNA with ORF expressing coat protein (CP, gray box). The genomic RNA is translated to produce p69 (hatched box) from initiation at AUG⁶⁹ and p206 (open box) from initiation at AUG²⁰⁶, but the CP ORF is silent (indicated by dashed outline). The ^m7GpppN cap of each RNA is indicated by an asterisk. The cruciform structure at the 3′ end represents the TLS. (B) Sequences and proposed conformation (9) of the largely unstructured 5′-UTR of TYMV genomic RNA. The initiation codons at nt 88 and 95 and corresponding translation products are marked. (C) Basic 5′-UTRs of LUC reporter RNA constructs. The sequence of the vector-derived (vec) 5′-UTR is shown, and the 5′ regions of paired constructs reporting initiation from each of the two TYMV AUG initiation codons are diagrammed. The wild-type LUC protein produced from the vec 5′-UTR begins with the dipeptide ME (single-letter amino acid code), as indicated. Constructs with 5′-TYMV sequences produce the LUC fusion proteins 69L or 206L, which have N-terminal fusions of 43 and 41 residues from the N-terminal portions of p69 and p206, respectively. The amino acid sequences derived from p69 and p206 are shown (single-letter code), along with two bridging residues derived from a PstI site used for cloning (LQ, in reverse shading). Reporter RNAs with the 69L or 206L 5′ sequences differ by the addition of a single residue just before the PstI site that functions to match the LUC coding sequences to the reading frame of the N-terminal fragment of p206 (unpublished data). (D) Basic 3′-UTRs of LUC reporter RNA constructs. The sequence of the vector-derived 19-nt 3′-UTR referred to as Bam is shown. This 3′-UTR is produced from DNA templates linearized with BamHI. The two types of TYMV 3′-UTR used are shown: TYg and TYsg, which include the 684-nt UTR of the genomic RNA and the 109-nt UTR of the subgenomic RNA, respectively. Both UTRs are fused as indicated to the generic Bam 3′-UTR sequence. Transcription from plasmid linearized with XmnI produces RNAs with the same 3′ termini as virion RNA.