Selection of the mRNA Translation Initiation Region by Escherichia coli Ribosomes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 17; pp. 6427 - 6431
• Main Authors: Calogero, Raffaele A., Pon, Cynthia L., Canonaco, Maria A., Gualerzi, Claudio O.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.09.1988; National Acad Sciences
• Subjects: Amino acids; Base Sequence; Biological and medical sciences; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genes, Synthetic; Kinetics; Messenger RNA; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Peptide initiation factors; Plasmids; Protein Biosynthesis; Reading frames; Ribosomes; Ribosomes - metabolism; RNA; RNA, Messenger - genetics; Transfer RNA; Translation. Translation factors. Protein processing; Messenger RNA; Ribosome; Escherichia coli; Translation initiation; Bacteria; Molecular interaction; Escherichieae; Mechanism; Enterobacteriaceae
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.17.6427

Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3′ end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka≈ 2 × 107 M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA.