Ribosomes: Structure, Function, and Dynamics

Cover......Page 1
Frontmatter......Page 2
Title Page......Page 3
Copright Page......Page 4
Preface......Page 6
Table of Contents......Page 8
Section I Ribosome structure......Page 11
1. Introduction......Page 13
2. Hibernating bears stimulated ribosome crystallization......Page 14
3. The ribosome is a polymerase......Page 16
4. Structural disorder with functional meaning......Page 18
6. Antibiotics targeting the ribosome: strategies, expectations and problems......Page 20
7. The ribosomal core is the optimized vestige of an ancient entity......Page 22
9. Future prospects......Page 23
References......Page 24
1. Introduction......Page 29
2.1. Insights from studies on the 30S subunit......Page 30
2.2. Structures of the ribosome with elongation factor Tu and aminoacyl tRNA......Page 32
3. Studies on termination......Page 33
5. Structure of a post-translocation complex of the ribosome with elongation factor G......Page 35
6. A structure of a ribosome recycling factor......Page 37
References......Page 38
1. Introduction......Page 41
2.1. The macrolides and chloramphenicol revisited......Page 42
2.2. The tuberactinomycins......Page 45
3.1. Regulation at the level of initiation: EF-P and the first peptide bond......Page 46
3.2. Nascent chains as regulators of protein synthesis: The TnaC leader peptide......Page 49
References......Page 51
2. How does the ribosome maintain the reading frame of messenger RNA during protein synthesis?......Page 55
2.2. Network of interactions of the ribosome with the mRNA downstream of the A-site codon......Page 56
2.4. Interactions of the ribosome with the mRNA region upstream of the P-site codon......Page 59
3. Stabilization of tRNA in the A site of the 70S ribosome......Page 61
References......Page 64
2.1. Genetics of T. thermophilus......Page 67
2.3. Streptomycin resistance and dependence......Page 68
2.4. Communication between ribosomal protein S12 and EF-Tu......Page 70
3.1. Probing the structural basis for streptomycin dependence......Page 71
3.2. Restructuring of the 30S subunit by rRNA modification......Page 72
References......Page 73
2.2. Ribosome positioning in polysomes......Page 75
2.3. Evidence for inter-ribosome regulation......Page 76
4.1. Translation initiation in bacteria......Page 77
4.2. A functional role for protein L9 in polysomes......Page 78
5. Conformational dynamics of ribosomes within polysomes......Page 80
References......Page 81
2. Overall view of the 80S yeast ribosome......Page 85
3. Ribosomal domain movements in the ratcheted state......Page 87
4. Rearrangement of ribosome functional sites upon ratcheting......Page 90
References......Page 91
1. Introduction......Page 93
2. The structure of the mammalian mitochondrial ribosome......Page 94
2.1. The small subunit of mammalian mitochondrial ribosomes......Page 96
2.3. Mammalian mitochondrial translation initiation factors......Page 97
2.4. Elongation factor G of mammalian mitochondria......Page 98
3.2. Large subunit of the Leishmania mitoribosome......Page 99
4. Structure of a chloroplast ribosome......Page 100
4.2. Large subunit of the chloroplast ribosome......Page 101
5. The polypeptide exit tunnels of the organellar ribosomes......Page 102
6. Conclusions......Page 103
References......Page 104
1. Introduction......Page 107
2. Modifications of rRNA stabilize ribosome structure......Page 108
3. Modifications in rRNA enhance ribosome interaction with ligands......Page 110
4. Modifications of rRNA unify ribosome interaction with ligands......Page 111
5. Modification of rRNA as an “assembly checkpoint”......Page 112
6. Modification of rRNA as a “quality mark” in ribosome assembly......Page 113
7. rRNA modification as antibiotic resistance mechanism......Page 115
8. rRNA modification and regulation of gene expression......Page 116
References......Page 117
Section II Recruiting the ribosome for translation......Page 121
1. Structure and function of bacterial translation initiation complexes......Page 123
1.1. Pre-initiation: mRNA binding and adaptation onto the 30S subunit......Page 124
1.2. Translation initiation factors......Page 126
1.3. The structure of translation initiation complexes......Page 128
2. The three-dimensional architecture of bacterial and eukaryotic polyribosomes......Page 130
3.1. Bacterial termination complexes with RF1 and RF2......Page 131
3.2. Bacterial termination complexes with the class-II release factor RF3......Page 134
References......Page 136
1. Introduction......Page 139
2. Roles of initiation factors in rapid and accurate formation of the 30S PIC......Page 140
3. Roles of initiation factors and initiator tRNA in subunit joining......Page 141
5. Alternative models of subunit joining and 70S IC formation......Page 144
5.1. Roles of GTP hydrolysis and Pi release in subunit joining......Page 145
5.2. IF2 interaction with the L12 protein of the 50S subunit promotes fast subunit joining......Page 146
6. Accuracy of initiator tRNA selection in initiation of mRNA translation......Page 147
8. IF2 mutants active in subunit joining in the absence of tRNA or GTP......Page 148
Acknowledgements......Page 149
References......Page 150
1. Background......Page 153
2. Nature and molecular basis of the cold-shock translational bias......Page 154
2.2. Trans-acting elements......Page 156
2.3. Multiple origin of the cold-shock translational bias......Page 159
2.4. Factors responsible for positive translational bias......Page 160
2.5. Factors responsible for negative translational bias......Page 161
3. Origin of the IFs/ribosome stoichiometric imbalance......Page 162
References......Page 163
1. Introduction......Page 165
2.1. eIF3......Page 166
2.2. eIF4E......Page 168
2.3. eIF4G......Page 169
2.4. eIF4A......Page 171
2.6. eIF4B......Page 172
2.8. PABP......Page 174
References......Page 175
1. Introduction......Page 181
2. Translation termination in eukaryotes......Page 182
3. Recycling of mammalian post-TCs by initiation factors at low Mg2+ concentrations......Page 183
4.1. ABCE1-mediated recycling of mammalian post-TCs......Page 185
4.2. The structure of ABCE1 and its relationship to function......Page 189
4.3. Evidence for the involvement of ABCE1 in translation termination and initiation......Page 190
4.4. Involvement of ABCE1 in cellular processes other than translation......Page 191
References......Page 192
Section III Decoding, fidelity, and peptidyl transfer......Page 197
2. Structures of the interface between EF-Tu and aa-tRNA......Page 199
3. Conformity and thermodynamic compensation......Page 201
4. How amino acid and tRNA specificity are achieved......Page 204
5. Estimating binding affinities and explaining T-stem sequence variation......Page 205
6. EF-Tu affinity and ribosomal function......Page 206
References......Page 207
2.2. Initial binding......Page 209
2.3. Codon recognition......Page 210
2.4. GTPase activation......Page 211
2.5. GTP hydrolysis by EF-Tu on the ribosome......Page 212
3.1. Error frequency......Page 213
3.2. Molecular mechanism of tRNA selection......Page 214
4.1. Structures of reaction intermediates......Page 216
4.2. Enzymology of the peptidyl transfer reaction......Page 217
References......Page 219
2.1. How does the recognition of sense codons work at a molecular level?......Page 223
2.2. How does the recognition of stop codons work at a molecular level?......Page 225
3.1. How is peptidyl transferase activity impacted by tRNA-mediated sense codon recognition?......Page 226
3.2. How is release activity impacted by RF1-mediated stop codon recognition?......Page 227
4.2. The pathway through the tRNA......Page 228
5. Does retrospective editing rely on the same signaling pathways?......Page 230
References......Page 232
1. Selection pressure on size, rate and accuracy of ribosomes in growing bacteria......Page 235
2.1. Early experiments on rate and accuracy......Page 237
2.2. More recent experiments on the accuracy of aminoacyl-tRNA selection......Page 238
3.1. Temperature dependence......Page 240
3.2. pH dependence1......Page 241
References......Page 244
1. Introduction......Page 247
2. Ribosomes defective in start codon selection......Page 249
3. Ribosomes defective in aminoacyl-tRNA selection......Page 250
4. Two screens for mutations that increase +1 frameshifting......Page 253
5.2. Certain mutations reported previously to decrease fidelity cause loss of function in the specialized ribosome system......Page 254
6. Concluding remarks......Page 255
References......Page 256
1. The ribosome as drug target: the issue of selectivity......Page 259
2.1. Chemistry......Page 260
2.2. Drug binding pocket......Page 261
2.4. Toxicity......Page 262
3. Establishment of a genetic model to study eukaryotic A-site function......Page 264
4. Aminoglycoside drug development......Page 267
References......Page 270
2.1. Ribosomal protein S5......Page 273
2.2. Experimental rationale and summary......Page 274
2.3. Isolation of mutations in protein S5......Page 275
2.7. RimJ can alleviate the fidelity defect associated with S5(G28D)......Page 276
2.9. The type of precursor sequence matters......Page 277
2.10. The paradox of the leader......Page 278
Section IV Elongation and ribosome dynamics......Page 281
2.1. Physical principles underlying fluorescence resonance energy transfer......Page 283
2.3. Design of donor-acceptor labeling schemes......Page 284
2.4. Total internal reflection fluorescence microscopy......Page 286
2.5. Limitations of single-molecule fluorescence microscopies......Page 288
2.6. Analysis of smFRET data......Page 289
3.1. Translocation of the mRNA-tRNA complex through the ribosome......Page 290
3.2. Structural rearrangements of the translational machinery important for translocation......Page 291
3.3. Identification and characterization of intermediate states connecting GS1 and GS2......Page 294
3.4. Allosteric regulation of the GS1/GS2 dynamic equilibrium......Page 295
3.5. EF-G-mediated control of the GS1/GS2 dynamic equilibrium during translocation......Page 297
4. Conclusions and future perspectives......Page 299
References......Page 300
2. Real-time translation in zero-mode waveguides......Page 305
3. Real-time monitoring of translation through sequential fluorescent-tRNA binding events......Page 307
4. Dynamic tRNA occupancy on the ribosome during translation......Page 308
5. Conclusion......Page 309
References......Page 311
1. Introduction......Page 313
2. Static states vs. ensembles of conformations......Page 314
3. Choosing the energy landscape reaction coordinate......Page 315
4. Further considerations in potential of mean force measurements......Page 317
5. The role of diffusion in energy landscapes: connecting kinetics and thermodynamics......Page 319
6. Energetic coupling between ribosomal compo nents......Page 320
7. Energy landscapes and X-ray crystallography......Page 321
9. Exploring energy landscapes through simulation......Page 324
References......Page 326
1. Introduction......Page 331
2. Ribosomal dynamics – complementary aspects explored by smFRET and cryo-EM......Page 332
3. mRNA-tRNA translocation......Page 333
References......Page 335
2.1. Spontaneous movement of tRNAs......Page 339
2.2. Kinetic mechanism of EF-G-dependent translocation......Page 341
3.1. The mechanism of ribosome recycling......Page 344
3.3. Ribosome recycling as in-vivo target of fusidic acid......Page 345
References......Page 346
1. Introduction......Page 349
2.1. Ensemble studies......Page 350
3. Enigmatic EF4(LepA)......Page 351
3.1. EF4 catalysis of partial back translocation......Page 352
3.3. A tentative model for the EF4 mechanism of action in vivo......Page 353
4.1. Ensemble stopped-flow experiments.......Page 354
5. EF-Tu interactions on the ribosome......Page 355
6. Future perspectives......Page 356
References......Page 357
2. Studies on the Mechanism of Translocation......Page 359
3. Studies on the Mechanism of Termination......Page 364
References......Page 369
1. Introduction......Page 371
2.1. tRNA-like domain......Page 372
3. SmpB structure and function......Page 373
3.3. SmpB-ribosome binding......Page 374
4.1. Empty A sites......Page 375
4.3. A-site cleavage......Page 376
5. Entering the A site of stalled ribosomes......Page 377
7.1. tmRNA determinants of frame selection......Page 378
8. Alternative rescue mechanisms......Page 379
References......Page 380
Section V Nascent peptide and tunnel interactions......Page 385
1. Introduction......Page 387
2.1. How diverse are the peptides that direct drug-dependent ribosome stalling?......Page 388
2.2. What is the length requirement of the nascent peptides that direct erythromycindependent ribosome stalling?......Page 390
2.3. What are the nascent peptide sequence requirements for ribosome stalling?......Page 391
3.1. What are the ribosomal sensors of the nascent peptide?......Page 393
3.2. What is broken in the stalled ribosome?......Page 396
4. What is the function of the antibiotic in drug-dependent ribosome stalling?......Page 397
ermBL and ermDL......Page 399
References......Page 400
1. An active role for the ribosomal tunnel during translation......Page 403
2.2. Interaction of the TnaC leader peptide with the ribosomal tunnel......Page 404
2.3. TnaC-mediated inactivation of the PTC......Page 406
2.4. TnaC leader peptide: Extended versus compacted......Page 407
3.1. Cryo-EM of translating ribosomes with nascent chains of high helical propensity......Page 408
3.2. Folding of helical peptides within distinct regions of the tunnel......Page 409
3.3. Characteristic behavior of helical peptides within the ribosomal tunnel......Page 410
4. Implications of interaction of nascent polypeptide chains within the ribosomal tunnel......Page 411
References......Page 412
2. The ribosomal tunnel......Page 415
3.1. Trigger factor......Page 417
3.2. Methionine aminopeptidase and peptide deformylase......Page 419
3.3. Signal recognition particle......Page 420
4. Membrane protein insertion......Page 422
4.1. Architecture of the SecYEG protein insertion pore......Page 423
4.2. Insertion of membrane proteins by YidC......Page 424
References......Page 425
Section VI Evolution......Page 429
1. Introduction......Page 431
2. Restrained evolution of the ribosome structure......Page 432
3. Dismantling the ribosome structure......Page 433
4. The origin of 23S rRNA......Page 435
5. Probabilistic quantification of the model......Page 436
7. The deteriorating evolution of mitochondrial ribosomes......Page 437
References......Page 438
Authors......Page 441