ИСТИНА

Plant alkaloid harringtonine (HT) was isolated in the 1960s from the bark of evergreen plant Cephalotaxus harringtonia grown in China. It belongs to the group of cephalotaxine derivatives, complex heterocyclic compounds having a strong anti-tumor effect. HT inhibits protein synthesis in eukaryotic cells. It is known from in vitro experiments that HT acts at the stage of elongation by binding to the A-site of the large ribosomal subunit and interfering with peptidyl transferase reaction. However, when added to cells, HT causes rapid polysome disassembly, because HT traps only de novo formed 80S complexes and is unable to bind to those ribosomes that are involved in productive synthesis of a polypeptide within the polysome. Thus, HT addition should lead to accumulation of arrested elongation complexes at the very beginning of coding region, presumably at the first (start) codon of the mRNA. Although this hypothesis has never been verified directly, this feature of HT was used recently in ribosome profiling experiments to map mRNA start codons in a genome-wide scale. We investigated the effect of HT on the elongation in a cell-free translation system using toe-printing technique. We found that HT poorly blocked elongation at the start codon of the beta-globin mRNA, but instead produced a very strong signal at the 9th codon encoded for Lys. Using a set of artificial mRNA constructs, we showed that HT specifically stalled elongating ribosomes in positions where their P-sites were occupied by either Lys, Arg or Tyr codons, while almost did not interfere with other amino acids. No influence of surrounding nucleotide or amino acid sequence, as well as no difference between synonymous codons, were detected. The same results were obtained with another cephalotaxine derivative, homoharringtonine. We assume that the amino acid specificity is dictated by a distinct architecture of the PTC occupied by peptidyl-tRNAs having either Lys, Arg or Tyr residues at the C-terminus of the nascent peptide. In this environment, HT either freezes the complex at pre-translocation step or blocks squeezing of the peptide chain into the ribosomal tunnel. These data reveal a rare example of an amino acid specific inhibition of elongation and extend our knowledge about mechanisms of action of PTC inhibitors.