Fig. 5.

Toeprinting shows that terminating ribosomes release from RNA in the presence of CHX but elongating ribosomes do not. In vitro synthesized, capped, and polyadenylated LUC reporter mRNA containing the wild-type AAP uORF was translated in the N. crassa CFTS in the presence of 10 µM (−) Arg or 2 mM (+) Arg (low or high Arg). CHX (final concentration 0.5 µg/µl) was added to the CFTS reactions either before the addition of mRNA (time T₀) or after translation was underway for 10 min (steady-state translation, time T₁₀). Steady-state reactions to which CHX was added were incubated in the presence of CHX for an additional 0, 2.5, or 5 min as indicated. The sequence of the mRNA can be directly deduced from the sequencing lanes reading from top to bottom. The positions of the uORF start and stop codons, and the LUC start codon, are boxed in the nucleotide sequence. The primer extension (toeprint) products corresponding to elongating ribosomes that have the uORF and LUC initiation codons in the P site are indicated by arrowheads; terminating ribosomes with the uORF termination codon in the A site are indicated with a star; and elongating ribosomes stalled within the uORF coding region are marked by a bracket. Control samples of RNA in reaction mixtures without extract and reaction mixtures containing extract but not mRNA are as indicated.