A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction.

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.

[1]  W. Wintermeyer,et al.  Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study. , 1983, Biochemistry.

[2]  Olivier Fayet,et al.  Recoding in bacteriophages and bacterial IS elements. , 2006, Trends in genetics : TIG.

[3]  Bernard Rees,et al.  Structural basis for messenger RNA movement on the ribosome , 2006, Nature.

[4]  R. Brimacombe,et al.  From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors. , 1995, RNA.

[5]  J. Hershey,et al.  The binding of fluorescein-labeled protein synthesis initiation factor 2 to Escherichia coli 30 S ribosomal subunits determined by fluorescence polarization. , 1982, The Journal of biological chemistry.

[6]  Jan van Duin,et al.  Translational initiation on structured messengers : another role for the Shine-Dalgarno interaction , 1994 .

[7]  R. Brimacombe,et al.  The location of mRNA in the ribosomal 30S initiation complex; site‐directed cross‐linking of mRNA analogues carrying several photo‐reactive labels simultaneously on either side of the AUG start codon. , 1991, EMBO Journal.

[8]  C. Gualerzi,et al.  Initiation of mRNA translation in prokaryotes. , 1990, Biochemistry.

[9]  J. Steitz,et al.  How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[10]  C. Gualerzi,et al.  Selection of the mRNA translation initiation region by Escherichia coli ribosomes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Ehrenberg,et al.  How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis. , 2006, Molecular cell.

[12]  R. Brimacombe,et al.  Prokaryotic translation: the interactive pathway leading to initiation. , 1994, Trends in genetics : TIG.

[13]  C. Vonrhein,et al.  Structure of the 30S ribosomal subunit , 2000, Nature.

[14]  C. Gualerzi,et al.  Translation initiation factor IF3: two domains, five functions, one mechanism? , 2001, The EMBO journal.

[15]  F. Schluenzen,et al.  Structure of Functionally Activated Small Ribosomal Subunit at 3.3 Å Resolution , 2000, Cell.

[16]  C. Gualerzi,et al.  Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors. , 1989, European journal of biochemistry.

[17]  F. Schluenzen,et al.  Structure of Functionally Activated Small Ribosomal Subunit , 2000 .

[18]  J. Shine,et al.  The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[19]  B. S. Laursen,et al.  Initiation of Protein Synthesis in Bacteria , 2005, Microbiology and Molecular Biology Reviews.

[20]  U Heinemann,et al.  Crystal structure of an RNA dodecamer containing the Escherichia coli Shine-Dalgarno sequence. , 1995, Journal of molecular biology.

[21]  Nan Yu,et al.  The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs , 2002, BMC Bioinformatics.

[22]  Neocles B. Leontis,et al.  Molecular modeling of nucleic acids , 1997 .

[23]  David A. Case,et al.  Modeling Unusual Nucleic Acid Structures , 1998 .

[24]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[25]  M. Santer,et al.  A single base change in the Shine-Dalgarno region of 16S rRNA of Escherichia coli affects translation of many proteins. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Harry F. Noller,et al.  The Path of Messenger RNA through the Ribosome , 2001, Cell.

[27]  S. Joseph,et al.  Unfolding of mRNA secondary structure by the bacterial translation initiation complex. , 2006, Molecular cell.

[28]  H. O. Voorma 26 Control of Translation Initiation in Prokaryotes , 1996 .

[29]  S Ringquist,et al.  Nature of the ribosomal mRNA track: analysis of ribosome-binding sites containing different sequences and secondary structures. , 1993, Biochemistry.

[30]  Marina V. Rodnina,et al.  Structural Basis for the Function of the Ribosomal L7/12 Stalk in Factor Binding and GTPase Activation , 2005, Cell.

[31]  H. Noller,et al.  Footprinting mRNA‐ribosome complexes with chemical probes. , 1994, The EMBO journal.

[32]  J. Holton,et al.  Structures of the Bacterial Ribosome at 3.5 Å Resolution , 2005, Science.

[33]  M. Selmer,et al.  Structure of the 70S Ribosome Complexed with mRNA and tRNA , 2006, Science.

[34]  Pascale Romby,et al.  Translational Operator of mRNA on the Ribosome: How Repressor Proteins Exclude Ribosome Binding , 2005, Science.

[35]  G D Stormo,et al.  High-level translation initiation. , 1990, Methods in enzymology.

[36]  C. Gualerzi,et al.  Initiation factors in the early events of mRNA translation in bacteria. , 2001, Cold Spring Harbor symposia on quantitative biology.

[37]  M. Ehrenberg,et al.  Novel roles for classical factors at the interface between translation termination and initiation. , 1999, Molecular cell.

[38]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[39]  Måns Ehrenberg,et al.  How initiation factors tune the rate of initiation of protein synthesis in bacteria , 2006, The EMBO journal.

[40]  V. Ramakrishnan,et al.  Insights into the decoding mechanism from recent ribosome structures. , 2003, Trends in biochemical sciences.

[41]  Jan van Duin,et al.  Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA. , 2003 .

[42]  V. Erdmann,et al.  Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit , 1989, Journal of bacteriology.

[43]  H. D. de Boer,et al.  Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[45]  I. Boni,et al.  Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation. , 2002, RNA.

[46]  Daniel N. Wilson,et al.  Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly. , 2005, Molecular cell.

[47]  V. Ramakrishnan,et al.  Crystal structure of an initiation factor bound to the 30S ribosomal subunit. , 2001, Science.