The effect of oligonucleotides complementary to the 3' end of the 16S rRNA on the formation of the bacterial 30S initiation complex

Abstract

Although much attention was focused on the role of the 16S RNA in mRNA selection by the 30S ribosomal subunit no true consensus site has emerged as yet. Oligonucleotides such as GAGG, UGAU and CCAA which are complementary to the 3' end of the 16S RNA stimulate the AUG-dependent binding of fMet-tRNA to 30S subunits. If those tetranucleotides are used in combination or if the octanucleotides GAGGUGAU and UGAUCCAA are applied, the degree of stimulation remains unchanged. Effects are strictly dependent on the presence of initiation factor 2 (IF-2) and cannot be produced by using A4 or U4. With sequences covalently linked to the AUG as in CCAAAUG and UGAUCCAAAUG, the efficiency of the initiation complex formation decreases significantly as compared to AUG with UGAUCCAAAUG being the least efficient mRNA analogue. The pentadecanucleotide GAGGUGAUCCAAAUG, however, shows the highest efficiency in directing the binding of the fMet-tRNA to 30S subunits and is clearly superior to AUG. Initiation factor 2 (IF-2), which stimulates tRNA binding significantly with AUG and CCAAAUG, both in terms of slope and plateau values of the binding curves, does not effect the initial rate of tRNA binding to GAGGUGAUCCAAAUG. In another set of experiments, where GAGG and AUG are separated by oligo(U) or oligo(A) sequences, the effect of chain length was investigated. mRNA analogues with a spacer of 6-9 nucleotides show the highest binding efficiencies, with a U spacer being superior to an A spacer, indicating that a more flexible spacer favours tRNA binding.