Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery

摘要

The ribosome is unique among Nature's biosynthetic machines in that the catalytic center is distinct from the substrate recognition pocket. This separation of catalysis and substrate binding suggests the ribosome may be uniquely well suited to manipulation for the synthesis of novel polymers. Indeed, in 1971, Rich and Fahnestock demonstrated that the protein biosynthetic machinery could synthesize polymers containing a random mixture of ester and amide linkages using mis-acylated hydroxy-Phe-tRNA. With the development of an efficient method for the chemoenzymatic synthesis of aa-tRNAs, several groups then used misacylated suppressor tRNAs or A/P-site substrates to test the ability of the protein biosynthetic machinery to accept backbone analogues, including α-hydroxy acids, N-methyl amino acids, α, α-disubstituted amino acids, β-amino acids, and D-amino acids. While many of these analogues could be incorporated in response to a stop codon, the yields for incorporation generally were low. Recently, we demonstrated that a sense codon reassignment strategy with a pure translation system allowed translation of multiple, adjacent sense codons with synthetic acyl-tRNA substrates. This approach and related approaches breaking codon degeneracy open the possibility of using the protein biosynthetic machinery for template encoded synthesis of novel backbone polymers of defined length and composition with a pool of synthetic acyl-tRNAs. The question, however, is whether the substrate tolerance and, in particular, poor yields reported previously for backbone analogues reflect use of a suppressor tRNA, which must compete with endogenous release factors for translation of a stop codon, processing by endogenous aa-tRNA synthetases or other metabolic enzymes, such as D-aa-deacylase, or the intrinsic specificity of the protein biosynthetic machinery. Here, as a further step toward polymer synthesis with sense codon reassignment, we determined the relative yields of peptides biosynthesized using a series of backbone analogues at a sense codon in a pure E. coli translation system (Figure 1). For efficient incorporation of any analogue, the acyl-tRNAs must be able to bind to EF-Tu, be delivered to the ribosome, and function well in both peptide bond formation and translocation.