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

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Abstract
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 phage R17-RNA-dependent binding of the initiator tRNA (fMet-tRNA) to 30-S ribosomal subunits. 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.cntdot.RNA hybrid acts as a positive control signal for the selection of the initiator tRNA by 30-S subunit.cntdot.mRNA complex. Tetranucleotides of the type A-U-G-N (where N = A, G, C or U) stimulate the IF-2[initiation factor-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, 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''-[.beta.,.gamma.-methylene]triphosphate 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.