Ribosome can resume the translation in both +1 or -1 frames after encountering an AGA cluster in Escherichia coli.
In Escherichia coli the rare codons AGG, AGA and CGA are reported to have a detrimental effect on protein synthesis, especially during the expression of heterologous proteins. In the present work, we have studied the impact of successive clusters of these rare codons on the accuracy of mRNA translation in E. coli. For this purpose, we have analyzed the expression of an mRNA which contains in its 3' region a triplet and a tandem of AGA codons. This mRNA is derived from the human hepatitis B virus (HBV) preC gene. Both in eukaryotic cells and in eukaryotic cell-free translation system, this mRNA, directs the synthesis of a single 25 kDa protein. However, in a conventional E. coli strain BL 21 (DE3), transformed with a plasmid expressing this protein the synthesis of four polypeptides ranging from 30 to 21.5 kDa can be observed. Using different approaches, notably expression of i) precore mutated proteins or ii) chimeric proteins containing HA- and Myc-tags downstream of the AGA clusters (respectively in the -1 or +1 frame), we have found that when the ribosome encounters the AGA clusters, it can then resume the translation in both +1 and -1 frames. This result is in agreement with the model proposed recently by Baranov et al. (Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10, 221-230), thus confirming that AGA/AGG codons can serve as sites for -1 frameshifting events. Only +1 frameshifting was suggested previously to occur at the AGA/AGG clusters.
Lainé
Sébastien
S
Laboratoire de Génétique et Biologie Cellulaire, UMR 81 59 CNRS/Université Versailles St-Quentin/EPHE, France.
Thouard
Anne
A
Komar
Anton A
AA
Rossignol
Jean-Michel
JM
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018965:Frameshifting, Ribosomal; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism
D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins