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Talk: Antibiotic inhibition of ribosome function (35 min)

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X Navigable Slide Index
  1. Introduction
  2. Protein synthesis
  3. Interference sites for protein synthesis
  4. David versus Goliath
  5. Drug targets on 16S rRNA
  6. 30S-antibiotic structure (1)
  7. 30S-antibiotic structure (2)
  8. Drug targets on 23S rRNA
  9. 50S-antibiotic structures
  10. Initiation inhibitors
  11. Kasugamycin history
  12. 30S-Kasugamycin structure
  13. Kasugamycin primary binding site (1)
  14. Kasugamycin primary binding site (2)
  15. Kasugamycin resistance
  16. Alternative conformations for helix 45
  17. Indirect resistance mechanism
  18. Kasugamycin secondary binding site
  19. Kasugamycin: 70S versus 30S
  20. Kasugamycin binds in mRNA pathway
  21. Kasugamycin inhibits tRNA binding to 30S
  22. Mechanism of kasugamycin action
  23. Inhibitors of initiation: edeine and pactamycin
  24. 30S-edeine structure
  25. 30S-pactamycin structure
  26. Pactamycin inhibits translocation
  27. Relative binding sites of initiation inhibitors
  28. Thiostrepton
  29. Chemical structure of thiostrepton
  30. Thiostrepton binding site on 23S rRNA
  31. 50S-thiostrepton structure
  32. 50S binding site of thiostrepton
  33. Thiostrepton interaction
  34. Native L11 position
  35. Free versus bound thiostrepton
  36. Minimal thiostrepton fragment
  37. Interaction of DHP
  38. Thiostrepton inhibits EF-G dependent Pi release
  39. Cryo-EM of 70S-EF-G complex
  40. Stalk base movement upon EF-G binding
  41. Thiostrepton allows initial binding of EF-G
  42. Thiostrepton prevents EF-G accommodation
  43. Thiostrepton resistance in bacteria
  44. Thiostrepton resistance in eukaryotes
  45. Peptidyltransferase (PTF) inhibitors
  46. Binding site of PTF inhibitors on 23S rRNA
  47. Inhibition of PTF activity
  48. Chemical structure of chloramphenicol
  49. 50S-chloramphenicol structure
  50. Peptidyltransferase centre
  51. Chloramphenicol binding site on 50S
  52. Chloramphenicol overlap with A-tRNA
  53. Chemical structures of macrolide antibiotics
  54. Macrolide binding site on 23S rRNA
  55. 50S-telithromycin structure
  56. Telithromycin binds in tunnel
  57. D50S-Tel versus H50S-Tel
  58. D50S-Tel versus D50S-Tel new
  59. New D50S-Tel versus H50S-Tel
  60. Species specific binding of antibiotics (1)
  61. Species-specific binding of antibiotics (2)
  62. Summary
  63. Bibliography
  64. END
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DETAILED SLIDE INDEX

  1. 1. Introduction
  2. 2. Protein synthesis
  3. 3. Interference sites for protein synthesis
  4. 4. David versus Goliath
  5. 5. Drug targets on 16S rRNA
  6. 6. 30S-antibiotic structure (1)
  7. 7. 30S-antibiotic structure (2)
  8. 8. Drug targets on 23S rRNA
  9. 9. 50S-antibiotic structures
  10. 10. Initiation inhibitors
  11. 11. Kasugamycin history
  12. 12. 30S-Kasugamycin structure
  13. 13. Kasugamycin primary binding site (1)
  14. 14. Kasugamycin primary binding site (2)
  15. 15. Kasugamycin resistance
  16. 16. Alternative conformations for helix 45
  17. 17. Indirect resistance mechanism
  18. 18. Kasugamycin secondary binding site
  19. 19. Kasugamycin: 70S versus 30S
  20. 20. Kasugamycin binds in mRNA pathway
  21. 21. Kasugamycin inhibits tRNA binding to 30S
  22. 22. Mechanism of kasugamycin action
  23. 23. Inhibitors of initiation: edeine and pactamycin
  24. 24. 30S-edeine structure
  25. 25. 30S-pactamycin structure
  26. 26. Pactamycin inhibits translocation
  27. 27. Relative binding sites of initiation inhibitors
  28. 28. Thiostrepton
  29. 29. Chemical structure of thiostrepton
  30. 30. Thiostrepton binding site on 23S rRNA
  31. 31. 50S-thiostrepton structure
  32. 32. 50S binding site of thiostrepton
  33. 33. Thiostrepton interaction
  34. 34. Native L11 position
  35. 35. Free versus bound thiostrepton
  36. 36. Minimal thiostrepton fragment
  37. 37. Interaction of DHP
  38. 38. Thiostrepton inhibits EF-G dependent Pi release
  39. 39. Cryo-EM of 70S-EF-G complex
  40. 40. Stalk base movement upon EF-G binding
  41. 41. Thiostrepton allows initial binding of EF-G
  42. 42. Thiostrepton prevents EF-G accommodation
  43. 43. Thiostrepton resistance in bacteria
  44. 44. Thiostrepton resistance in eukaryotes
  45. 45. Peptidyltransferase (PTF) inhibitors
  46. 46. Binding site of PTF inhibitors on 23S rRNA
  47. 47. Inhibition of PTF activity
  48. 48. Chemical structure of chloramphenicol
  49. 49. 50S-chloramphenicol structure
  50. 50. Peptidyltransferase centre
  51. 51. Chloramphenicol binding site on 50S
  52. 52. Chloramphenicol overlap with A-tRNA
  53. 53. Chemical structures of macrolide antibiotics
  54. 54. Macrolide binding site on 23S rRNA
  55. 55. 50S-telithromycin structure
  56. 56. Telithromycin binds in tunnel
  57. 57. D50S-Tel versus H50S-Tel
  58. 58. D50S-Tel versus D50S-Tel new
  59. 59. New D50S-Tel versus H50S-Tel
  60. 60. Species specific binding of antibiotics (1)
  61. 61. Species-specific binding of antibiotics (2)
  62. 62. Summary
  63. 63. Bibliography
  64. 64. END

RELATED TALKS

Play 'The interaction of antibiotics with the large ribosomal subunit from Haloarcula marismortui'
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TOPICS COVERED IN THIS TALK

  • Introduction to a paradox: how can a universally conserved cellular component be a target for pharmacologically useful antibiotics?
  • Introduction to ribosome structure
  • Antibiotic binding sites cluster in the ribosome
  • The source of the species specificity of antibiotics that interact with the A-site cleft
  • How macrolides interact with the ribosome
  • The structural consequences of mutations that make ribosomes resistant to macrolides
  • The toxicity of macrolides is not well understood
  • 13-deoxytedanolide, a novel macrolide with novel species specificity that binds to a novel site in the ribosome
Play 'Antibiotics that target the 50S ribosome subunit: resistance and other considerations'
Antibiotics that target the 50S ribosome subunit: resistance and other considerations More info
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TOPICS COVERED IN THIS TALK

  • Why target the 50S ribosomal subunit?
  • Analysis of antibacterial market
  • Mechanisms of drug resistance
  • Sequestration
  • How do antibiotics gain entry into cells
  • Porins
  • Lack of uptake
  • Self-promoted uptake drug modification
  • Target modifications
  • Drug efflux
  • Major superfamilies of transport proteins
Play 'The structure of the intact ribosome and ribosomal subunit interactions'
The structure of the intact ribosome and ribosomal subunit interactions More info
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TOPICS COVERED IN THIS TALK

  • Introduction to the architecture of the bacterial ribosome
  • Ribosome composition and landmarks
  • Ribosomal subunit interface
  • Conformational changes in the ribosome
Play 'Structural insights into decoding of mRNA by the ribosome'
Structural insights into decoding of mRNA by the ribosome More info
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TOPICS COVERED IN THIS TALK

  • tRNA: the central player in translation
  • Selectivity and transition state
  • Decoding: selection of the correct tRNA
  • The bacterial ribosome
  • Antibiotics change the fidelity of the ribosome
  • Selection of aa-tRNA by the ribosome
  • The atomic structure of the 30S subunit
  • Testing the decoding model
  • Minor groove recognition of base pairing geometry
  • Tautomers and base pairs
  • Streptomycin
  • Base pairing and fidelity
Play 'Translocation: movement of tRNA and mRNA through the ribosome'
Translocation: movement of tRNA and mRNA through the ribosome More info
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TOPICS COVERED IN THIS TALK

  • Ribosomes and translation
  • Molecular composition of the ribosome
  • Why is the ribosome so big?
  • The mRNA channel in the 30S subunit
  • Positions of tRNA in the A-, P- and E-sites
  • Structural dynamics of the ribosome during translocation
  • The hybrid states elongation cycle
  • tRNA binding during its movement through the ribosome
  • The 70S ribosome
  • Distortion of tRNA structure by the ribosome
  • The ratchet model
  • Cryo-EM experiment
  • Inferred relationship between ratcheting and hybrid states
  • FRET changes
  • Intersubunit movement
  • Testing the ratchet model for translocation
  • Translocation assay
  • What defines the mechanism of translocation
  • Sparsomycin
  • The energetics of translocation
  • E-site tRNA
  • The ribosome is a molecular machine

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TALK'S CITATION

Wilson, D. (2008), "Antibiotic inhibition of ribosome function", in Steitz, T. (ed.), The Mechanisms of Ribosome Function: Insights into protein synthesis, The Biomedical & Life Sciences Collection, Henry Stewart Talks Ltd, London (online at http://hstalks.com/?t=BL0321580)

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ABOUT THIS TALK

Speaker(s)

Dr. Daniel Wilson Show Biography

SPEAKER BIOGRAPHY

Dr. Daniel Wilson – Ludwig-Maximilians-Universitat Munchen,Germany

Daniel N. Wilson is an independent group leader at the Gene Center and Department of Chemistry and Biochemistry, LMU Munich. He received his undergraduate training at Otago University in New Zealand investigating translational recoding, and prior to joining the LMU in 2007, conducted postdoctoral work in the ribosome crystallography group at the Max-Planck Institute for Molecular Genetics in Berlin, Germany. One of Daniel's current areas of interest is the mechanism by which antibiotics inhibit the translational apparatus as well as how ribosomes gain resistance to antibiotic action.

Publication Date

May, 2008

Topics Covered

Overview of antibiotics that target the ribosome... more

TOPICS COVERED IN THIS TALK

  • Overview of antibiotics that target the ribosome
  • Different mechanisms by which translation initiation inhibitors, such as kasugamycin and edeine, perturb initiator tRNA binding
  • Structural insight into how the thiopeptide class of antibiotics, such as thiostrepton and micrococcin, inhibit stable binding of translation factors, such as EF-G, to the ribosome
  • Mechanism by which drugs, such as chloramphenicol and macrolides inhibit the peptidyltransferase reaction
  • How species specific differences in antibiotic interaction sites between different organisms can have a dramatic influence on drug binding

Series

The Mechanisms of Ribosome Function

OTHER TALKS IN THIS SERIES

INTRODUCTION TO THE RIBOSOME
Play '1. The principle of translation'
1. The principle of translation More info
Prof. Anders Liljas

TOPICS COVERED IN THIS TALK

  • The understanding of heredity and translation of proteins based on the genetic information
  • Intensive structural studies of ribosomes, tRNAs and translation factors
  • A historical overview
  • Main components of translation
ARCHITECTURE OF THE RIBOSOME RNA-PROTEIN MACHINE ASSEMBLY
Play '2. High resolution structure of ribosomal subunits RNA/RNA and protein/RNA interactions'
2. High resolution structure of ribosomal subunits RNA/RNA and protein/RNA interactions More info
Prof. Nenad Ban

TOPICS COVERED IN THIS TALK

  • X-ray crystallography
  • Protein synthesis
  • High resolution structure of ribosomal subunits
  • RNA motifs and interactions
  • RNA protein interactions
  • Metal ions stabilizing RNA structure
  • Functional implications of RNA/RNA and RNA/protein interactions during protein synthesis
Play '3. The structure of the intact ribosome and ribosomal subunit interactions'
3. The structure of the intact ribosome and ribosomal subunit interactions More info
Dr. Jamie H. Doudna Cate

TOPICS COVERED IN THIS TALK

  • Introduction to the architecture of the bacterial ribosome
  • Ribosome composition and landmarks
  • Ribosomal subunit interface
  • Conformational changes in the ribosome
Play '4. Assembly of the 30S ribosomal subunit in vitro and in cells'
4. Assembly of the 30S ribosomal subunit in vitro and in cells More info
Prof. James Williamson

TOPICS COVERED IN THIS TALK

  • Bacterial ribosome biogenesis
  • The Nomura assembly map
  • RNA folding and protein binding
  • Single molecule fluorescence
  • The basis for cooperative assembly
  • Isotope pulse chase experiments
  • Protein binding kinetics
  • The assembly landscape
DECODING AND PEPTIDE BOND FORMATION
Play '5. How aminoacyl-tRNA synthetases translate the genetic code'
5. How aminoacyl-tRNA synthetases translate the genetic code More info
Dr. Stephen Cusack

TOPICS COVERED IN THIS TALK

  • Fidelity of protein synthesis
  • Architecture of aminoacyl-tRNA synthetase
  • ATP and amino acid recognition by the two classes of synthetases
  • The activation reaction
  • Cognate tRNA recognition by synthetases
  • The aminoacylation reaction
  • Direct and indirect routes to aminoacylation
  • Error correction (editing) by aminoacyl-tRNA synthetases
  • Aminoacyl-tRNA synthetases as antibiotic targets
Play '6. Structural insights into decoding of mRNA by the ribosome'
6. Structural insights into decoding of mRNA by the ribosome More info
Prof. Venki Ramakrishnan

TOPICS COVERED IN THIS TALK

  • tRNA: the central player in translation
  • Selectivity and transition state
  • Decoding: selection of the correct tRNA
  • The bacterial ribosome
  • Antibiotics change the fidelity of the ribosome
  • Selection of aa-tRNA by the ribosome
  • The atomic structure of the 30S subunit
  • Testing the decoding model
  • Minor groove recognition of base pairing geometry
  • Tautomers and base pairs
  • Streptomycin
  • Base pairing and fidelity
Play '7. The structural basis for how the large ribosomal subunit catalyses peptide bond formation'
7. The structural basis for how the large ribosomal subunit catalyses peptide bond formation More info
Prof. Thomas Steitz

TOPICS COVERED IN THIS TALK

  • Overview of protein synthesis by the ribosome
  • Nature of the polypeptide exit tunnel in the large subunit
  • The ribosome is a ribozyme
  • Substrate induced fit at the peptidyl transferase center
  • Proton shuttle mechanism and transition state stabilization
  • Substrate interactions at the large subunit E-site
  • Movie of the peptidyl transferase reaction
Play '8. Chemistry of peptide bond formation'
8. Chemistry of peptide bond formation More info
Prof. Rachel Green

TOPICS COVERED IN THIS TALK

  • Fundamental processes during protein synthesis
  • Peptidyl transfer catalysis by RNA-rich active site
  • Ribosome structure solution provided new insights
  • Peptide bond formation and peptide release
  • Active site of the ribosome
  • Universally conserved nucleotides of the active site
  • Affinity tag purification of mutant ribosomes
  • Active site mutations and rates of peptidyl transfer
  • A-site substrates: tRNA vs. puromycin
  • "Uninduced" state of ribosome and P-site substrate
  • A-site substrates-induced conformational changes
  • Peptide release activity of variant ribosomes
  • RF catalysis in the large subunit
  • RF conserved amino acids in bacteria
  • Consequences of RF1 site-directed mutations
  • Nucleophile partitioning
  • Mutants that lost water specificity
  • RF components functioning in activating hydrolysis
  • Regulation of translation post-initiation
Play '9. Elongation of Protein Synthesis: Structural Basis of the Process of Decoding'
9. Elongation of Protein Synthesis: Structural Basis of the Process of Decoding More info
Prof. Marina Rodnina

TOPICS COVERED IN THIS TALK

  • Fidelity in biological processes
  • Elongation step of protein synthesis
  • Ribosome structures
  • Errors in protein synthesis
  • The decoding problem
  • Kinetic proof-reading
  • Kinetic model of tRNA selection
  • Role of induced fit in tRNA selection
  • Recognition of codon-anticodon complexes by the 30S ribosomal subunit
  • Local and global conformational changes of the 30S subunit
  • Interactions of tRNA during decoding
  • Active role of tRNA
  • Accommodation of tRNA in the A site
INITIATION OF PROTEIN SYNTHESIS
Play '10. The structure-based mechanism of translation initiation in bacteria and eukaryotes'
10. The structure-based mechanism of translation initiation in bacteria and eukaryotes More info
Prof. Jeffrey Kieft

TOPICS COVERED IN THIS TALK

  • Importance, principles and pathways of translation initiation
  • Components of initiation in bacteria
  • The Shine-Dalgarno sequence
  • Structures and function of bacterial initiation factors
  • Complexity of eukaryotes initiation
  • Cap recognition and regulation
  • Structures of eukaryotic initiation factors
Play '11. Factor independent initiation of protein synthesis by IRES RNAs'
11. Factor independent initiation of protein synthesis by IRES RNAs More info
Prof. Jeffrey Kieft

TOPICS COVERED IN THIS TALK

  • Translation initiation in eukaryotes: two pathways
  • IRES-driven translation
  • Types of IRESes
  • IRES manipulation of ribosomes
  • Folding and structure of the IGR IRESes
  • IRES interactions with the ribosome and molecular mimicry
  • New IRES mechanistic models
  • Towards an understanding of more complex IRESes
ELONGATION AND TERMINATION OF PROTEIN SYNTHESIS
Play '12. Structural insights into aminoacyl-tRNA delivery by EF-Tu and translocation by EF-G'
12. Structural insights into aminoacyl-tRNA delivery by EF-Tu and translocation by EF-G More info
Prof. Joachim Frank

TOPICS COVERED IN THIS TALK

  • Schematic of elongation cycle
  • Role of elongation factors EF-G and EF-Tu
  • Molecular mimicry of elongation factors
  • Binding positions of factors and tRNA during elongation cycle
  • Process of tRNA selection
  • Proofreading
  • Role of bases A1492 and A1493
  • GTP hydrolysis on EF-Tu
  • tRNA as a molecular spring
  • Translocation
  • Intersubunit ratchet motion
  • Induced fit
  • FRET studies of ratchet motion
  • Hybrid states of tRNA
  • Role of GTP hydrolysis in translocation
Play '13. Translocation: movement of tRNA and mRNA through the ribosome'
13. Translocation: movement of tRNA and mRNA through the ribosome More info
Prof. Harry Noller

TOPICS COVERED IN THIS TALK

  • Ribosomes and translation
  • Molecular composition of the ribosome
  • Why is the ribosome so big?
  • The mRNA channel in the 30S subunit
  • Positions of tRNA in the A-, P- and E-sites
  • Structural dynamics of the ribosome during translocation
  • The hybrid states elongation cycle
  • tRNA binding during its movement through the ribosome
  • The 70S ribosome
  • Distortion of tRNA structure by the ribosome
  • The ratchet model
  • Cryo-EM experiment
  • Inferred relationship between ratcheting and hybrid states
  • FRET changes
  • Intersubunit movement
  • Testing the ratchet model for translocation
  • Translocation assay
  • What defines the mechanism of translocation
  • Sparsomycin
  • The energetics of translocation
  • E-site tRNA
  • The ribosome is a molecular machine
Play '14. Recoding: getting more out of the message by shifting reading frame and redefining codon meaning'
14. Recoding: getting more out of the message by shifting reading frame and redefining codon meaning More info
Prof. John Atkins
Prof. Raymond Gesteland

TOPICS COVERED IN THIS TALK

  • Dynamic reprogramming of genetic readout
  • 3 broad types
  • Change in linearity, i.e. programmed ribosomal frame shifting
  • Codon redefinition, i.e. commonly "stop codons" specifying an amino acid, a standard one or selenocysteine
  • Discontiguity
CO-TRANSLATIONAL PROTEIN SECRETION
Play '15. Mechanism of translocon function: current insights and models'
15. Mechanism of translocon function: current insights and models More info
Prof. Arnold Driessen

TOPICS COVERED IN THIS TALK

  • Bacterial protein sorting
  • Evolutionary conserved translocons
  • Structure of SecYEG complex
  • Oligomeric assemblies of the SecYEG complex
  • Oligomeric state of the active SecYEG complex
  • Orientation of the SecYEG in dimeric complexes
  • Active or passive role of ribosome and SecA during channel opening
HOW ANTIBIOTICS TARGET THE RIBOSOME
Play '16. Antibiotics that target the 50S ribosome subunit: resistance and other considerations'
16. Antibiotics that target the 50S ribosome subunit: resistance and other considerations More info
Dr. Joyce Sutcliffe

TOPICS COVERED IN THIS TALK

  • Why target the 50S ribosomal subunit?
  • Analysis of antibacterial market
  • Mechanisms of drug resistance
  • Sequestration
  • How do antibiotics gain entry into cells
  • Porins
  • Lack of uptake
  • Self-promoted uptake drug modification
  • Target modifications
  • Drug efflux
  • Major superfamilies of transport proteins
Play '17. The interaction of antibiotics with the large ribosomal subunit from Haloarcula marismortui'
17. The interaction of antibiotics with the large ribosomal subunit from Haloarcula marismortui More info
Prof. Peter Moore

TOPICS COVERED IN THIS TALK

  • Introduction to a paradox: how can a universally conserved cellular component be a target for pharmacologically useful antibiotics?
  • Introduction to ribosome structure
  • Antibiotic binding sites cluster in the ribosome
  • The source of the species specificity of antibiotics that interact with the A-site cleft
  • How macrolides interact with the ribosome
  • The structural consequences of mutations that make ribosomes resistant to macrolides
  • The toxicity of macrolides is not well understood
  • 13-deoxytedanolide, a novel macrolide with novel species specificity that binds to a novel site in the ribosome
Now Playing
18. Antibiotic inhibition of ribosome function
Dr. Daniel Wilson
Play '19. Structure-based drug design targeting infectious disease'
19. Structure-based drug design targeting infectious disease More info
Dr. Erin Duffy

TOPICS COVERED IN THIS TALK

  • Many antibiotics inhibit the ribosome
  • Intelligent engine builds perpetual pipeline
  • Crystallography shows 50S is a target-of-targets
  • From new X-ray crystals to new leads
  • Analog(Trade Mark)
  • LINKER essentials: tethering two binding features
  • BOMB essentials
  • MUSIC essentials: core-swapping
  • QikProp: calculating drug-like properties
  • Structures + Analog(Trade Mark): balanced compounds
  • Case study: enhanced oxazolidinones
  • Structural foundations
  • Path to enhanced oxazolidinones
  • Pi contributions represent key interactions
  • Probing selectivity of sparsomycin
  • Seeking a new lead
  • Difference density map showing rib-X prototype
  • Perfect match
  • A minimal scaffold for lead optimization
  • Minimal scaffold binds effectively in A-site
  • Activity against H. influenzae
  • H. influenzae activity with time evolution
  • H. flu model delivers differentiable oxazolidinones
  • Moderate in vivo activity measured
  • Predictive model for rat oral bioavailability
  • First pool of drug candidates
  • Structures + predictive models = time-saving filters

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