The Ribosome Discriminates the Structure of the Amino Acid at its Peptidyl-Transferase Center

180 Pages·2011·5.71 MB·English

The Ribosome Discriminates the Structure of
the Amino Acid at its Peptidyl-Transferase
Center

Michael Thomas Englander

Submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
under the Executive Committee
of the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY
2011
2011
Michael Thomas Englander
All Rights Reserved
Abstract
The Ribosome Discriminates the Structure of the Amino Acid at its
Peptidyl-Transferase Center

Michael Thomas Englander

Contemporary interpretations of Crick’s original Adaptor Hypothesis view both the amino
acid and the tRNA body as passive participants during aminoacyl-tRNA (aa-tRNA)
selection by the ribosome. Recent experimental evidence investigating tRNA mutants that
miscode due to mutations that lie outside the anticodon as well as data from the unnatural
amino acid mutagenesis field that shows that different unnatural amino acids, when
esterified to the same suppressor tRNA, produce different amounts of protein, suggest
that aa-tRNA selection is considerably more complex than originally envisioned in
Crick’s Adaptor Hypothesis and that aa-tRNA selection may extend to the amino acid
itself. Here, using D-aa-tRNAs as substrates on natural, fully modified tRNAs and a
highly purified in vitro translation system, we demonstrate that D-aa-tRNAs inhibit
catalysis at the ribosomal peptidyl-transferase center (PTC), substantiating our hypothesis
that the ribosome is in fact sensitive to the structure of the amino acid.
The implications of these results affects multiple disciplines. First, we provide a
rationale for the impaired incorporation of unnatural amino acids widely observed in the
unnatural amino acid mutagenesis field and demonstrate that D-amino acids can be
incorporated into protein using the ribosome. Second, we show that D-aa-tRNAs inhibit
translation at a previously undetected mechanistic step in the elongation cycle of protein
synthesis that occurs after incorporation into peptide. This step presumably arises from
the presence of the D-amino acid at the C-terminus of the nascent peptide which impedes
structural rearrangements of the PTC that normally partition the PTC between an active
and inactive conformation.
Perhaps most significantly, the incorporation of D-amino acids into protein is a
plausible mechanism that might underlie human disease states. Analagous to editing
defective synthetases that mischarge natural amino acids in vivo resulting in protein
misfolding phenotypes, we predict that the aminoacylation of D-amino acids and
subsequent incorporation into proteins by the ribosome in vivo would lead to similar
defects, thereby explaining the near universal conservation of D-aminoacyl-tRNA
deacylase enzymes.
Table of Contents
Table of Contents ............................................................................................................... i
List of Figures ..................................................................................................................... v
List of Tables ................................................................................................................... viii
Acknowledgements .......................................................................................................... ix
Dedication ........................................................................................................................... x
Chapter 1 Introduction ...................................................................................................... 1
1.1 The Central Dogma and the Fidelity of Protein Synthesis ................................... 1
1.2 The Adaptor Hypothesis ....................................................................................... 4
1.3 The Ribosome and the Contemporary Framework of the Elongation Cycle of
Protein Synthesis ................................................................................................... 5
1.3.1 Aminoacyl-tRNA Selection .......................................................................... 7
1.3.2 Peptide Bond Formation ............................................................................... 9
1.3.3 Translocation .............................................................................................. 13
1.4 Challenges to Contemporary Interpretations of the Adaptor Hypothesis ............. 14
1.4.1 The tRNA .................................................................................................... 15
1.4.2 The Amino Acid ......................................................................................... 18
1.5 Conclusions ........................................................................................................... 25
1.6 References ............................................................................................................. 26
Chapter 2 Experiments with Amino Acid Backbone Analogues ................................. 35
2.1 Background ........................................................................................................... 35
2.2 Limitations to Previous Methodologies ................................................................ 36
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2.3 The Position of a Methyl Backbone Substituent Affects Different Steps of the
Elongation Cycle of Protein Synthesis ............................................................... 37
2.4 Future Directions .................................................................................................. 40
2.5 References ........................................................................................................... 41
Chapter 3 The Ribosome Discriminates the Structure of the Amino Acid at its
Peptidyl-Transferase Center ........................................................................................... 43
3.1 New Methods ........................................................................................................ 43
3.1.1 Ribozyme Misacylation of Natural tRNAs ................................................. 43
3.1.2 Electrophoretic Thin Layer Chromatography ............................................. 45
3.2 D-Amino Acids Exhibit Translation Disorders During the Elongation Cycle ..... 45
3.2.1 Dipeptide Synthesis Translations ............................................................... 47
3.2.2 Tripeptide Synthesis Translations ............................................................... 49
3.2.3 Tetrapeptide Synthesis Translation ............................................................. 51
3.3 Racemization Controls .......................................................................................... 52
3.4 D-Amino Acids Inhibit Catalysis in the PTC ....................................................... 54
3.5 Generality of D-amino Acid PTC Inhibition ........................................................ 57
3.6 Conclusions ........................................................................................................... 69
3.7 References ........................................................................................................... 74
Chapter 4 The Enforcement of Homochirality ............................................................. 79
4.1 Implications of In Vitro Results .......................................................................... 79
4.2 Cellular Defense Network for the Enforcement of Homochirality In Vivo ........ 83
4.3 Future Directions ................................................................................................ 88
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4.4 References ........................................................................................................... 89
Chapter 5 Methods .......................................................................................................... 93
5.1 Ribosomes, Translation Factors, tRNA Synthetases, tRNAs and mRNAs .......... 93
5.2 tRNA Aminoacylation .......................................................................................... 95
5.3 Buffer System ................................................................................................... 103
5.4 In-Vitro Translations ......................................................................................... 104
5.4.1 70S Initiation Complex Formation ........................................................... 104
5.4.2 Ternary Complex Formation and EF-G .................................................... 104
5.4.3 Di- and Tripeptide Assays ........................................................................ 105
5.4.4 Tetrapeptide Assay ................................................................................... 106
5.4.5 Toeprinting Assay ..................................................................................... 106
5.4.6 Nitrocellulose Filter Binding Assay ......................................................... 109
5.4.7 Puromycin Reactivity ............................................................................... 110
5.5 Generation of ∆dadA and ∆dtd Single Deletion Strains ..................................... 111
5.6 Generation of ∆dadA and ∆dtd Double Deletion Strain ..................................... 114
5.6.1 Preparation of Donor Lysate ..................................................................... 115
5.6.2 Transduction ............................................................................................. 115
5.7 References ........................................................................................................... 119
Appendix ......................................................................................................................... 121
A.1 Ribosome purification ........................................................................................ 121
A.2 Tev Protease, EF-Tu, EF-Ts and EF-G Protein Purifications ............................ 130
A.3 T7 RNA Polymerase Purification .................................................................... 134
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A.4 E. Coli CCA Enzyme Purification ................................................................... 137
A.5 Ribozyme Preparation (eFx & dFx) ................................................................. 141
A.6 aaRS Aminoacylation Reactions ...................................................................... 148
A.7 Analysis of aaRS Aminoacylation Reaction ...................................................... 150
A.7.1 FPLC ........................................................................................................ 150
A.7.2 Acid PAGE Gels ...................................................................................... 151
A.8 Labeling of tRNA with α-32P-ATP ................................................................... 152
A.9 Ribozyme Aminoacylation Reaction ................................................................. 153
A.10 TLC Analysis of Ribozyme Aminoacylation Efficiency ............................... 154
A.11 In Vitro Transcriptions ................................................................................... 154
A.12 Translation Reaction Protocol .......................................................................... 156
A.12.1 Translation Reaction (for eTLC) ........................................................... 157
A.12.2 Filter Binding ......................................................................................... 158
A.12.3 Toeprinting Exceptions .......................................................................... 158
A.12.4 Puromycin Reactivity ............................................................................ 160
A.12.5 Buffers ................................................................................................... 160
A.13 Total mRNA Purification and RT-PCR ........................................................... 161
A.14 Plasmid Preps, Linearization and Mutagenesis ............................................. 164
A.15 References ...................................................................................................... 166

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List of Figures
Figure 1.1 The Central Dogma ........................................................................................... 3
Figure 1.2 The E. coli 70S Ribosome ................................................................................. 6
Figure 1.3 Elongation cycle of protein synthesis ................................................................ 6
Figure 1.4 Kinetic proofreading and the mechanism of aa-tRNA selection ....................... 8
Figure 1.5 Conformational rearrangements in the PTC .................................................... 10
Figure 1.6 rRNA nucleotides in the PTC .......................................................................... 11
Figure 1.7 The tRNA is distorted in the A/T state ............................................................. 17
Figure 1.8 Unnatural amino acid mutagenesis .................................................................. 19
Figure 2.1 Incorporation of amino acid backbone analogues ........................................... 35
Figure 2.2 Subtle modifications to the amino acid backbone effect different stages of the
elongation cycle ................................................................................................................. 38
Figure 2.3 Summary of HPLC traces ................................................................................ 39
Figure 3.1 Ribozymes and substrates ................................................................................ 44
Figure 3.2 Experimental set-up and eTLC analysis .......................................................... 46
Figure 3.3 Dipeptide synthesis translation ........................................................................ 48
Figure 3.4 EF-Tu dependence of fMet-D-Phe dipeptide formation .................................. 48
Figure 3.5 Tripeptide synthesis translation ....................................................................... 50
Figure 3.6 fMet-D-Phe-Lys tripeptide synthesis rate and endpoint are independent of
increased concentration of the third amino acid, Lys-tRNALys ......................................... 50
Figure 3.7 Tetrapeptide synthesis translation ................................................................... 52
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Figure 3.8 Tripeptide synthesis translation products co-migrate with authentic marker
peptides using HPLC analysis ........................................................................................... 53
Figure 3.9 D-Phe-tRNAPhe inhibits catalysis in the PTC .................................................. 56
Figure 3.10 D-Val-tRNAVal and D-Lys-tRNALys dipeptide synthesis translations ........... 58
Figure 3.11 D-Val-tRNAVal and D-Lys-tRNALys exhibit translation disorders ................ 59
Figure 3.12 Tripeptide synthesis translation products using D-Val-tRNAVal and
D-Lys-tRNALys co-migrate with authentic marker peptides using HPLC analysis ........... 60
Figure 3.13 Tetrapeptide synthesis translation of fMet-D-Lys-Phe-Glu .......................... 61
Figure 3.14 Increased concentration of EF-G or Phe-tRNAPhe does not effect the endpoint
of the fMet-D-Lys-Phe tripeptide synthesis reaction ......................................................... 62
Figure 3.15 Peptidyl-D-Val-tRNAVal triggers a proofreading mechanism in which a
fraction of ribosomes larger than that observed with peptidyl-D-Phe-tRNAPhe partitions
into a non-productive state ................................................................................................. 63
Figure 3.16 Peptidyl-D-Lys-tRNALys triggers a proofreading mechanism in which a
fraction of ribosomes smaller than that observed with peptidyl-D-Phe-tRNAPhe partitions
into a non-productive state ................................................................................................. 64
Figure 3.17 The fraction of translationally stalled ribosomes depends on the side chain of
the D-amino acid ................................................................................................................ 66
Figure 3.18 Structural models of the PTC containing analogs of either a
peptidyl-L-aa-tRNA or a peptidyl-D-aa-tRNA at the P site .............................................. 67
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