Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers

Abstract

Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNA Phe, tRNA fMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517–528, 1397–1404, and 1534–1542. The binding of cDNAs 1534–1541 and 1398–1403 decreased in the presence of the ligand pair poly(U)/tRNA Phe. Only the binding of cDNA 1534–1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNA Phe or tRNA fMet alone. The poly(AGU)/tRNA fMet ligand pair caused an additional decline in the binding of cDNA 1534–1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517–528 or 1398–1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534–1541 in the presence of mRNAs or tRNA. At the 517–528 and 1398–1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers. The oligomers complementary to 16S bases 517–528 and 1398–1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [1].