• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Oct 1990; 87(19): 7668–7672.
PMCID: PMC54809

Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis.

Abstract

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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (961K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. [PubMed]
  • de Smit MH, van Duin J. Control of prokaryotic translational initiation by mRNA secondary structure. Prog Nucleic Acid Res Mol Biol. 1990;38:1–35. [PubMed]
  • Hall MN, Gabay J, Débarbouillé M, Schwartz M. A role for mRNA secondary structure in the control of translation initiation. Nature. 1982 Feb 18;295(5850):616–618. [PubMed]
  • Buell G, Schulz MF, Selzer G, Chollet A, Movva NR, Semon D, Escanez S, Kawashima E. Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C (IGF-I). Nucleic Acids Res. 1985 Mar 25;13(6):1923–1938. [PMC free article] [PubMed]
  • Tessier LH, Sondermeyer P, Faure T, Dreyer D, Benavente A, Villeval D, Courtney M, Lecocq JP. The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli. Nucleic Acids Res. 1984 Oct 25;12(20):7663–7675. [PMC free article] [PubMed]
  • Schmidt BF, Berkhout B, Overbeek GP, van Strien A, van Duin J. Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2. J Mol Biol. 1987 Jun 5;195(3):505–516. [PubMed]
  • Spanjaard RA, van Dijk MC, Turion AJ, van Duin J. Expression of the rat interferon-alpha 1 gene in Escherichia coli controlled by the secondary structure of the translation-initiation region. Gene. 1989 Aug 15;80(2):345–351. [PubMed]
  • Tomich CS, Olson ER, Olsen MK, Kaytes PS, Rockenbach SK, Hatzenbuhler NT. Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E. coli. Nucleic Acids Res. 1989 Apr 25;17(8):3179–3197. [PMC free article] [PubMed]
  • Skripkin EA, Adhin MR, de Smit MH, van Duin J. Secondary structure of the central region of bacteriophage MS2 RNA. Conservation and biological significance. J Mol Biol. 1990 Jan 20;211(2):447–463. [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. [PubMed]
  • Remaut E, Stanssens P, Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. [PubMed]
  • Devos R, van Emmelo J, Contreras R, Fiers W. Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA. J Mol Biol. 1979 Mar 15;128(4):595–619. [PubMed]
  • Berkhout B, Schmidt BF, van Strien A, van Boom J, van Westrenen J, van Duin J. Lysis gene of bacteriophage MS2 is activated by translation termination at the overlapping coat gene. J Mol Biol. 1987 Jun 5;195(3):517–524. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Angenent GC, Posthumus E, Bol JF. Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2. Virology. 1989 Nov;173(1):68–76. [PubMed]
  • Freier SM, Kierzek R, Jaeger JA, Sugimoto N, Caruthers MH, Neilson T, Turner DH. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. [PMC free article] [PubMed]
  • Gualerzi C, Risuleo G, Pon CL. Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3. Biochemistry. 1977 Apr 19;16(8):1684–1689. [PubMed]
  • Ellis S, Conway TW. Initial velocity kinetic analysis of 30 S initiation complex formation in an in vitro translation system derived from Escherichia coli. J Biol Chem. 1984 Jun 25;259(12):7607–7614. [PubMed]
  • Forchhammer J, Lindahl L. Growth rate of polypeptide chains as a function of the cell growth rate in a mutant of Escherichia coli 15. J Mol Biol. 1971 Feb 14;55(3):563–568. [PubMed]
  • Gouy M, Grantham R. Polypeptide elongation and tRNA cycling in Escherichia coli: a dynamic approach. FEBS Lett. 1980 Jun 30;115(2):151–155. [PubMed]
  • Calogero RA, Pon CL, Canonaco MA, Gualerzi CO. Selection of the mRNA translation initiation region by Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6427–6431. [PMC free article] [PubMed]
  • Iserentant D, Fiers W. Secondary structure of mRNA and efficiency of translation initiation. Gene. 1980 Apr;9(1-2):1–12. [PubMed]
  • Selker E, Yanofsky C. Nucleotide sequence of the trpC-trpB intercistronic region from Salmonella typhimurium. J Mol Biol. 1979 May 15;130(2):135–143. [PubMed]
  • Queen C, Rosenberg M. Differential translation efficiency explains discoordinate expression of the galactose operon. Cell. 1981 Jul;25(1):241–249. [PubMed]
  • Looman AC, Bodlaender J, Comstock LJ, Eaton D, Jhurani P, de Boer HA, van Knippenberg PH. Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 1987 Aug;6(8):2489–2492. [PMC free article] [PubMed]
  • Munson LM, Stormo GD, Niece RL, Reznikoff WS. lacZ translation initiation mutations. J Mol Biol. 1984 Aug 25;177(4):663–683. [PubMed]
  • Thomas JO, Szer W. RNA-helix-destabilizing proteins. Prog Nucleic Acid Res Mol Biol. 1982;27:157–187. [PubMed]
  • Subramanian AR. Structure and functions of ribosomal protein S1. Prog Nucleic Acid Res Mol Biol. 1983;28:101–142. [PubMed]
  • Roberts MW, Rabinowitz JC. The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes. J Biol Chem. 1989 Feb 5;264(4):2228–2235. [PubMed]
  • Rosa MD. Structure analysis of three T7 late mRNA ribosome binding sites. J Mol Biol. 1981 Mar 25;147(1):55–71. [PubMed]
  • Jaeger JA, Turner DH, Zuker M. Improved predictions of secondary structures for RNA. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7706–7710. [PMC free article] [PubMed]
  • Scherer GF, Walkinshaw MD, Arnott S, Morré DJ. The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments. Nucleic Acids Res. 1980 Sep 11;8(17):3895–3907. [PMC free article] [PubMed]
  • Stormo GD, Schneider TD, Gold LM. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. [PMC free article] [PubMed]
  • Stormo GD, Schneider TD, Gold L, Ehrenfeucht A. Use of the 'Perceptron' algorithm to distinguish translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2997–3011. [PMC free article] [PubMed]
  • Schneider TD, Stormo GD, Gold L, Ehrenfeucht A. Information content of binding sites on nucleotide sequences. J Mol Biol. 1986 Apr 5;188(3):415–431. [PubMed]
  • Dreyfus M. What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs? J Mol Biol. 1988 Nov 5;204(1):79–94. [PubMed]
  • Precup J, Parker J. Missense misreading of asparagine codons as a function of codon identity and context. J Biol Chem. 1987 Aug 15;262(23):11351–11355. [PubMed]
  • Ganoza MC, Kofoid EC, Marlière P, Louis BG. Potential secondary structure at translation-initiation sites. Nucleic Acids Res. 1987 Jan 12;15(1):345–360. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...