Tetranucleotides as Effectors for the Binding of Initiator tRNA to Escherichia coli Ribosomes

Oligonucleotides such as G‐A‐G‐G, which are complementary to the C‐U‐C‐C region at the 3′ end of 16‐S RNA, inhibit the R17‐RNA‐dependent binding of the initiator tRNA (fMet‐tRNA) to 30‐S ribosomal subunits. However, if phage RNA is replaced by A‐U‐G, the same oligonucleotides stimulate the binding of fMet‐tRNA to the 30‐S subunits. This indicates that the formation of the RNA · RNA hybrid acts as a positive control signal for the selection of the initiator tRNA by the 30‐S‐subunit · mRNA complex. Tetranucleotides of the type A‐U‐G‐N (where N = A, G, C or U) stimulate the IF‐2‐dependent binding of fMet‐tRNA to the 30‐S subunit more effectively than A‐U‐G, with A‐U‐G‐R better than A‐U‐G‐Y (where R is a purine nucleoside and Y is a pyrimidine nucleoside). Since the 3′‐terminal adenosine in A‐U‐G‐A can be replaced by 6‐deamino‐adenosine, a stacking type of interaction between U‐33 of tRNA and N of A‐U‐G‐N should additionally stabilize the codon‐anticodon complex. The situation is strictly reversed for 70‐S ribosomes where A‐U‐G is the best codon followed by A‐U‐G‐U, A‐U‐G‐C, A‐U‐G‐G and A‐U‐G‐A. Replacement of GTP by guanosine 5′‐[β, γ‐methylene]triphosphate (Guo PP [CH 2 ] P ] results in A‐U‐G‐A becoming more efficient than A‐U‐G as the codon for the binding of fMet‐tRNA to 70‐S ribosomes. This indicates that IF‐2 and GTP hold the anticodon of the fMet‐tRNA in a conformation capable of binding to a tetranucleotide codon. GTP hydrolysis and release of IF‐2 from the 70‐S ribosome results in a change of the tertiary structure of fMet‐tRNA as a consequence of which the initiator tRNA reassumes the conformation which preferentially binds to A‐U‐G.