Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis.
We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region. The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo. The results reveal a strict correlation between translational efficiency and the stability of the helix. An increase in its delta G0 of -1.4 kcal/mol (i.e., less than the difference between an A.U and a G.C pair) corresponds to the reduction by a factor of 10 in initiation rate. Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match. We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition. Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region. Ribosomes appear not to recognize nucleotides outside the Shine-Dalgarno region and the initiation codon.
de Smit
M H
MH
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
van Duin
J
J
D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D002213:Capsid / Q000235:genetics*; D003090:Coliphages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D012270:Ribosomes / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D015678:Viral Structural Proteins