Adenine-containing codons enhance protein synthesis by promoting mRNA binding to ribosomal 30S subunits provided that specific tRNAs are not exhausted

Abstract

Adenines downstream of the initiation codon promote protein synthesis; however, some adenine-containing codons (AGA, AGG and AUA) at early positions inhibit protein synthesis when cognate tRNA is exhausted. It has also been reported, although not convincingly, the presence of adenines enhancing mRNA binding to the ribosome. To understand these apparent inconsistencies we analyzed the effect of these codons in mRNA-ribosome binding strength, mRNA stability, the production of peptidyl-tRNA (pep-tRNA) and protein synthesis. Constructs harboring lacZ derivatives were obtained by site directed mutagenesis where tandems of GGG, AGG, AGA, ATA and AAA codons were inserted at codon positions 2–3 and 3–4. Codons containing more adenines, irrespective of being common or rare, (AAA, ATA and AGA) promoted a higher synthesis of β-galactosidase (β-gal) in comparison with those rich in guanines (GGG and AGG) in a wild type transcription-translation system. Full-length mRNAs were also detected when the adenine-rich constructs were expressed in wild type cells. Under conditions where the pool of tRNAs is readily exhausted (pep-tRNA hydrolase defective cells), the adenine-rich lacZ derivatives caused a stronger and general inhibition of protein synthesis and cell growth. With the exception of the ATA lacZ derivative, only plasmid constructs containing hungry codons generated pep-tRNA (AGA and to a lesser extent AGG) in Pth defective cells. Codons containing more adenines clearly promoted lacZ mRNA binding to 30S subunit. The GGG lacZ mRNA showed a moderate increase in binding when mRNA secondary structures were disrupted by heating mRNAs before the binding assay which agrees with the lacZ mRNA secondary structures predicted with MFOLD. Altogether, these results indicate that mRNA binding to ribosome plays a major role in the enhancement of translation by adenine-rich codons irrespective of codon usage. This effect is naturally expressed in wild type systems and depends on adenine content, in contrast to the inhibition caused after over-expressing the lacZ derivatives containing rare codons in Pth defective cells.