70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Significance Until now, two initiation modes for bacterial translation have been described: (i) the standard 30S-binding mode, where the small ribosomal subunit selects the initiation site on an mRNA with the help of three initiation factors (IFs), and (ii) the rare initiation of leaderless mRNAs, which are mRNAs carrying the initiation AUG within the first 5 nt at the 5′-end. The existence of a third “70S-scanning” mode for bacterial initiation was conjectured in past decades but has remained experimentally unproven. Here, we demonstrate the existence of a 70S-scanning mode of initiation and characterize its mechanistic features. The three initiation modes demonstrate specific patterns of requirements for IF1 and IF3. According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1–IF3). Here, we describe a novel type of initiation termed “70S-scanning initiation,” where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine–Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.

[1]  J W Hershey,et al.  Translation initiation factor IF1 is essential for cell viability in Escherichia coli , 1994, Journal of bacteriology.

[2]  J. Frank,et al.  Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits. , 2007, Journal of molecular biology.

[3]  Ignacio Tinoco,et al.  The Ribosome Uses Two Active Mechanisms to Unwind mRNA During Translation , 2011, Nature.

[4]  E. Goldman,et al.  Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli , 1984, Journal of bacteriology.

[5]  C. Gualerzi,et al.  Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs , 1999, Molecular microbiology.

[6]  A. Teleman,et al.  DENR•MCT-1 Promotes Translation Reinitiation Downstream of uORFs to Control Tissue Growth , 2014, Nature.

[7]  N. Blüthgen,et al.  Molecular Systems Biology 9; Article number 675; doi:10.1038/msb.2013.32 Citation: Molecular Systems Biology 9:675 , 2022 .

[8]  Shashi Bhushan,et al.  SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center , 2011, PLoS biology.

[9]  J Frank,et al.  Location of translational initiation factor IF3 on the small ribosomal subunit. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  K. Nierhaus,et al.  How the ribosome moves along the mRNA during protein synthesis. , 1994, The Journal of biological chemistry.

[11]  A. Haenni,et al.  The behaviour of acetylphenylalanyl soluble ribonucleic acid in polyphenylalanine synthesis. , 1966, Biochimica et biophysica acta.

[12]  Sotaro Uemura,et al.  Heterogeneous Pathways and Timing of Factor Departure during Translation Initiation , 2012, Nature.

[13]  A. Bogdanov,et al.  Unusual ribosome binding properties of mRNA encoding bacteriophage λ repressor , 1992 .

[14]  Isabella Moll,et al.  Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs. , 2004, Nucleic acids research.

[15]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[16]  C. Hellen,et al.  Reinitiation and other unconventional posttermination events during eukaryotic translation. , 2013, Molecular cell.

[17]  M. Nomura,et al.  Post-transcriptional regulation of the str operon in Escherichia coli. Ribosomal protein S7 inhibits coupled translation of S7 but not its independent translation. , 1994, Journal of molecular biology.

[18]  V. Mootha,et al.  Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans , 2009, Proceedings of the National Academy of Sciences.

[19]  M. Deutscher,et al.  A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN. , 1984, The Journal of biological chemistry.

[20]  L. Gold,et al.  Selection of the initiator tRNA by Escherichia coli initiation factors. , 1989, Genes & development.

[21]  R. Kaempfer,et al.  Initiation factor IF-3: a specific inhibitor of ribosomal subunit association. , 1972, Journal of molecular biology.

[22]  A Danchin,et al.  Toward an understanding of the formylation of initiator tRNA methionine in prokaryotic protein synthesis. II. A two-state model for the 70S ribosome. , 1976, Biochemistry.

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

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

[25]  C L Pon,et al.  Mechanism of protein biosynthesis in prokaryotic cells , 1984, FEBS letters.

[26]  S. Marzi,et al.  A structural view of translation initiation in bacteria , 2009, Cellular and Molecular Life Sciences.

[27]  G. Stent,et al.  Intracellular condition of Escherichia coli transfer RNA. , 1966, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Pon,et al.  Specificity and properties of the destabilization, induced by initiation factor IF-3, of ternary complexes of the 30-S ribosomal subunit, aminoacyl-tRNA and polynucleotides. , 1976, European journal of biochemistry.

[29]  Rachel Green,et al.  Dom34 Rescues Ribosomes in 3′ Untranslated Regions , 2014, Cell.

[30]  A Yonath,et al.  Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3 , 2001, The EMBO journal.

[31]  A. Kaji,et al.  The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits. , 2005, RNA.

[32]  J. Elf,et al.  Selective charging of tRNA isoacceptors induced by amino‐acid starvation , 2005, EMBO reports.

[33]  J. F. Atkins,et al.  A dual-luciferase reporter system for studying recoding signals. , 1998, RNA.

[34]  M. Rodnina,et al.  The ribosome‐bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex , 2010, EMBO reports.

[35]  A. Kaji,et al.  The ribosome-recycling step: consensus or controversy? , 2006, Trends in biochemical sciences.

[36]  Daniel N. Wilson,et al.  Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG , 2008, Proceedings of the National Academy of Sciences.

[37]  T. Ueda,et al.  Spermidine inhibits transient and stable ribosome subunit dissociation , 2006, FEBS letters.

[38]  Takuya Ueda,et al.  Protein synthesis by pure translation systems. , 2005, Methods.

[39]  Måns Ehrenberg,et al.  Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G. , 2005, Molecular cell.

[40]  J. van Duin,et al.  Scanning model for translational reinitiation in eubacteria. , 1990, Journal of molecular biology.

[41]  Takeshi Yokoyama,et al.  Structural insights into initial and intermediate steps of the ribosome‐recycling process , 2012, The EMBO journal.

[42]  R. Kaempfer.  Ribosomal subunit exchange during protein synthesis. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[43]  K. Manchester.  Resolution with a protein synthesising system of conflicting estimates for the stability constant for KATP3- formation. , 1980, Biochimica et biophysica acta.

[44]  M. Ehrenberg,et al.  Termination of translation: interplay of mRNA, rRNAs and release factors? , 2003, The EMBO journal.

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

[46]  Michael C. Jewett,et al.  Protein synthesis by ribosomes with tethered subunits , 2015, Nature.

[47]  C. Gualerzi,et al.  Structure and function of bacterial initiation factors. , 2002, Current protein & peptide science.

[48]  Frank Dieterich,et al.  Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica. , 2004, FEMS microbiology letters.

[49]  M. Rodnina,et al.  Sequence of steps in ribosome recycling as defined by kinetic analysis. , 2005, Molecular cell.

[50]  Tanel Tenson,et al.  Evolutionary and genetic analyses of mitochondrial translation initiation factors identify the missing mitochondrial IF3 in S. cerevisiae , 2012, Nucleic acids research.

[51]  G. Zurawski,et al.  Structure of the Escherichia coli S10 ribosomal protein operon. , 1985, Nucleic acids research.

[52]  Joachim Frank,et al.  The Cryo-EM Structure of a Translation Initiation Complex from Escherichia coli , 2005, Cell.

[53]  Christian M T Spahn,et al.  Codon-Anticodon Interaction at the P Site Is a Prerequisite for tRNA Interaction with the Small Ribosomal Subunit* , 2002, The Journal of Biological Chemistry.

[54]  H. Noller,et al.  How the ribosome hands the A-site tRNA to the P site during EF-G–catalyzed translocation , 2014, Science.

[55]  A. Bogdanov,et al.  Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor. , 1992, Nucleic Acids Research.

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

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

[58]  H. Noller,et al.  Interaction of translation initiation factor 3 with the 30S ribosomal subunit. , 2001, Molecular cell.

[59]  Pohl Milón,et al.  Kinetic control of translation initiation in bacteria , 2012, Critical reviews in biochemistry and molecular biology.

[60]  C. Yanofsky,et al.  Nonsense codons and polarity in the tryptophan operon. , 1966, Journal of molecular biology.

[61]  M Grunberg-Manago,et al.  Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes. , 1975, Journal of molecular biology.

[62]  Hiroyuki Furusawa,et al.  70 S Ribosomes Bind to Shine–Dalgarno Sequences without Required Dissociations , 2008, Chembiochem : a European journal of chemical biology.

[63]  Koichi Ito,et al.  Transient idling of posttermination ribosomes ready to reinitiate protein synthesis. , 2004, Biochimie.

[64]  J. Hershey,et al.  Initiation factor and ribosome levels are coordinately controlled in Escherichia coli growing at different rates. , 1983, The Journal of biological chemistry.

[65]  Y. Shimizu,et al.  Evidence for the Translation Initiation of Leaderless mRNAs by the Intact 70 S Ribosome without Its Dissociation into Subunits in Eubacteria* , 2004, Journal of Biological Chemistry.

[66]  G. Blaha,et al.  Formation of 70S ribosomes: large activation energy is required for the adaptation of exclusively the small ribosomal subunit. , 2002, Biophysical chemistry.

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

[68]  H. Noller,et al.  mRNA Helicase Activity of the Ribosome , 2005, Cell.