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. 2014 Mar 5;289(17):11739–11754. doi: 10.1074/jbc.M114.554030

FIGURE 7.

FIGURE 7.

Reinitiation at downstream AUGs depends on the Kozak context and translation of the upstream ORF down to the UAA in the stop/start site. A, sequences represent the 5′ region of the VP2 coding frame. Top, the wild type (wt) sequence is given with the stop/start site underlined, and the VP2 start codon is shown in boldface. Above the sequence, codon numbers are given with codon 1 representing the VP2 start codon in the WT sequence. Below, mutants with a 2-nt substitution of the start codon and introduction of a downstream AUG, which is highlighted in boldface and underlined, are shown. The position of the codon of the VP2 frame that is mutated to AUG is given on the right side of the mutant sequences (expressed as codon number with the VP2 AUG representing codon 1). In construct pNV52b, two further mutations were introduced to create a bad Kozak context of the downstream AUG. B, gels with the proteins precipitated after transient expression of the indicated constructs (antisera as described for Fig. 3). A protein precipitated by V5 antibody that is smaller than VP2-V5, detected in addition to the long protein and VP2t-V5, is marked by an asterisk. C, nucleotide and encoded amino acid sequence of the stop/start region of the WT construct (top) and of the region equivalent to the upper row of the WT sequence of the frameshift (FS) construct containing an inserted C residue leading to premature termination of Pep6-VP1 translation (inserted C in boldface and stop codon underlined). Below these two sequences, the sequence context around the stop/start region of the tested start site mutants is given (see A). Please note that for each start site mutant, two versions were tested, one with the WT Pep6-VP1 coding sequence and one with the frameshift insertion. D, gels with the proteins precipitated after transient expression of the indicated constructs (antisera as described for Fig. 3). A protein precipitated by V5 antibody that is smaller than VP2-V5, detected in addition to the long protein and VP2t-V5, is marked by an asterisk. Lanes FS show products expressed from the frameshift versions of the indicated constructs. Vacc, vaccinia virus.