TOWARDS a CONSISTENT CHRONOLOGY TO EXPLAIN THE EVOLUTION OF THE RIBOSOME

TOWARDS A CONSISTENT CHRONOLOGY TO EXPLAIN THE EVOLUTION OF THE RIBOSOME     ZHANG BO (B.SCI.,USTC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN COMPUTATION & SYSTEMS BIOLOGY (CSB) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2012   DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously Zhang Bo ____________________ Digitally signed by Zhang Bo DN: cn=Zhang Bo, o, ou, email=primrosebo33@gmail.com, c=US Date: 2013.06.06 21:28:21 +08'00' ZHANG BO 24th Aug. 2012       II   Acknowledgements It was not possible for me to realize the great support I have gotten from my friends and family until I finished my thesis and looked back over the journey past. They have helped and continually supported me along this long and fulfilling road. I would like to express my great thanks to my PhD supervisor, Professor Christopher W. V. Hogue, who is not only a mentor but also a dear friend. Throughout the four years study, I have been confused and lost my directions. I could not reach where I am today without his inspirational, supportive, kind and patient guidance, and editorial assistance in preparing this thesis. Many thanks go to my MIT-Singapore program co-advisor, Professor Gil Alterovitz, who provided encouraging and instructive comments about my projects and showed me great kindness when I was studying in MIT. A good support system is important in surviving and staying in graduate school. I am very grateful to my department, Singapore-MIT Alliance, for providing me four years Graduate Scholarship financial assistance. I am also grateful to our co-chair, Professor Gong Zhiyuan and former co-chair, Professor Hew Choy Leong and the staff and students in SMA, especially in Computation & Systems Biology. I also have to thank the members of my PhD committee and my examiners for their helpful advice and suggestions in general. I am so lucky to have been surrounded by wonderful colleagues. I will take this opportunity to thank all my workmates and lab mates who have     III   contributed to such a pleasant environment for the past four years: Shweta Ramdas who contributed to this project in her honors year; Zhao Chen, a wonderful friend; Liao Xuanhao who provided a great help in the wet lab and all my lab mates. I am sincerely grateful that I have this group of passionate people to work with in Hogue’s lab. I could always ask for advice and help. And Kootala Parasuraman Sowmya, our secretary, is always there for us. Also essential to my thesis were the software and applications, especially the Design Structure Matrix software developed by Loomeo. I will also thank a group of experts who helped keep my thesis real. They have given me the permission to include their beautiful and accurate figures in my thesis. I especially thank my mom and dad. They have sacrificed so much in their lives for my comfortable life and provided me unconditional love and care. I would not make this real without their support. I truly thank Li Qiushi for always standing by my side and sharing my dreams.       IV   Table of Contents Acknowledgements . III   Table of Contents . V   Summary VII   List of Figures IX   List of Tables . XI   Chapter Introduction .   1.1  Background  and  Significance   .  5   1.1.1  Ribosomal  Structure  and  Function    6   1.1.2  The  RNA  World  Theory  and  Other  Origin  Hypotheses    15   1.1.3  Hydrothermal  Vents    22   1.1.4  Current  Research  on  the  Evolutionary  Timeline  of  the  Ribosome    26   1.2  Objectives  and  Proposed  Solutions   .  52   1.2.1  Objectives  and  Specific  Aims    52   1.2.2  Research  Scope   .  56   Chapter Material and Methods 58   2.1  Chronology  Models  for  E.  coli  Ribosome   .  58   2.1.1  Preparation  for  the  Chronology  Models   .  58   2.1.2  The  Chronology  Models    60   2.2  Chronology  Models  for  the  E.  coli  Ribosome  -­‐  DSM    65   2.2.1  Design  Structure  Matrix  (DSM)   .  65   2.2.2  Domain  Mapping  Matrix  (DMM)    70   2.3  Visualization  of  the  Ribosomal  Evolution  Chronology   .  71   2.3.1  Video  of  Hydrothermal  Vent   .  72   2.3.2  Animation  of  Molecular  Structures   .  73   2.3.3  Animation  of  Molecular  Structures  -­‐  Software   .  74   2.4  Simplest  Proto-­‐Ribosome    75   2.5  Overview  of  Joint  DSM  Model  Development  Approach    76   Chapter Chronological Evolution of E. coli Ribosomal LSU . 84   3.1  DSM  Chronology  Models  for  LSU   .  84       V   3.1.1  “Proteins-­‐earliest”  Model   .  85   3.1.2  Hybrid  Model    96   3.1.3  Discussion  of  the  Optimal  and  Sub-­‐Optimal  Paths   .  100   3.2  Polypeptidyl-­‐Transferase  Center  (PTC)    103   3.3  Theory  in  the  Ribosomal  Evolution  between  Archaea  and  Bacteria    106   3.4  Discussion  for  the  Evolutionary  Model  of  LSU   .  111   Chapter Chronological Evolution of E. coli Ribosomal SSU . 117   4.1  Preliminary  Data  for  SSU    117   4.1.1  A-­‐minor  Interactions   .  118   4.1.2  Protein-­‐Protein  Interactions   .  120   4.2  Banded  Chronology  DSMs  Model  of  the  SSU   .  122   4.3  Discussion  of  the  Banded  DSM  Model  of  the  SSU   .  129   Chapter Chronological Evolution of the E. coli Ribosome 136   5.1  Inter-­‐Subunit  Interface    136   5.2  Joint  Chronology  of  the  SSU  and  LSU   .  138   5.3  Discussion  of  the  Joint  Chronology  of  the  E.  coli  Ribosome    149   Chapter Animation of the Ribosomal Evolution . 152   6.1  Visualization  of  the  Joint  Chronology  Model   .  152   6.2  Hydrothermal  Vent  System  Background    157   6.3  Animation  of  the  Joint  Chronology  of  the  Ribosome   .  158   6.4  Discussion  of  the  Animation  Process   .  158   Chapter The Structure of the Simplest Proto-Ribosome . 161   7.1  Three  Cores    161   7.2  Discussion  of  the  Proposed  r29-­‐PTC  Proto-­‐Ribosome  Model    167   Chapter Conclusion and Future Research 169   Bibliography 173   Appendices . 186       VI   Summary The ribosome comprises the structure and mechanism for the translation of nucleic acid gene sequences into proteins in all living creatures. The large subunit (LSU) of the ribosome is reducible to an ancient catalytic core peptidyl-transferase structure (PTC) (Agmon, Bashan et al. 2005). A model of hierarchical addition of E. coli 23S (where ‘S’ refers to the Sedimentation Coefficient) rRNA modular inserts (HIM) was proposed (Bokov and Steinberg 2009) explaining how inserts led from the PTC to the full ribosome. Based on this information, a detailed chronology of the ribosome was developed, including rRNA modules and ribosomal proteins (rproteins) in the large and small subunits (SSU) of E. coli using the Design Structure Matrix (DSM), and employing dependencies from 3D structure and topology. The DSM does not use sequence information, yet the results are remarkably well validated against other models of ribosomal evolution. The earliest period of structure accumulation is better fitted to a protein-free assembly than a protein-early model. For the first two proteins appearing in the chronology, L22c is the beta-strand protrusion of L22 and L32 binds via a bare alpha helix next to L22c in a crevice proximal to the polypeptide exit tunnel. These are congruent with a theory that the first proteins were simple units of secondary structure, prior to the evolution of folded forms. A feedback loop from these two crevices may provide selective pressure for fixation of initially random sequences for stronger binding forms that may have streamlined nascent peptide exit. Such feedback could have helped fix the earliest portion of the genetic code. While there is no L32 in the archaea, part     VII   of the space occupied by L32 was found filled with a structure arising from a sequence insert into archaeal L22 that may have displaced L32 from the archaeal ribosome. Decomposition of the SSU 3D structure into rRNA module inserts reveals two originating cores labeled r23 and r29. The r29 module is consistent with a functional form of the earliest proto-SSU and its structure validated by a new reduced mitochondrial SSU sequence. A banded DSM chronology shows how the SSU may have evolved in stages from these two core structures. The interface between the LSU and SSU together with the 5S fragment and all r-proteins were combined together into a final DSM of the entire E. coli ribosome, which was iteratively refined by constructing full animations of the chronology in the Maya software package. Docking supports a potential functional form of the earliest proto-ribosome comprising the PTC and r29, suggesting that the SSU and LSU co-evolved from the start. The chronology supports a transition from mini-tRNA to full-tRNA upon the build-up of the subunit interface, a period congruent with the fixation of the genetic code, and a last common ribosomal ancestor structure before the split of archaea and bacteria. With the 2D and 3D illustrations of the evolutionary process presenting the ribosomal chronology, the results represent the most complete story of ribosomal evolution so far presented.       VIII   List of Figures Figure 1.1 Structure of intact E. coli 70S ribosome. . 11   Figure 1.2 Ribosome architecture in prokaryotes and eukaryotes. . 12   Figure 1.3 Overview of the bacterial translation. 13   Figure 1.4 Timeline of evolution. . 18   Figure 1.5 RNA reactor from a hydrothermal vent pore network. . 24   Figure 1.6 Evolutionary transition of mini-tRNA to full-length tRNA. . 32   Figure 1.7 The symmetrial RNA dimer structures of PTC. 44   Figure 1.8 Hierarchical model of the LSU from Bokov and Steinberg. . 47   Figure 1.9 Secondary and tertiary structure of the SSU. 48   Figure 1.10 Onion-like model. 50   Figure 2.1 A brief introduction to the Design Structure Matrix (DSM). 67   Figure 2.2 LOOMEO SSU input structures. . 68   Figure 2.3 Domain Mapping Matrix structures in the LOOMEO. . 70   Figure 2.4 Domain mapping graph. 71   Figure 2.5 Project DSM analysis stages. 77   Figure 3.1 Interaction networks. . 84   Figure 3.2 Domain Mapping Matrix for the LSU. 88   Figure 3.3 DSM of modules and proteins insertion order. . 89   Figure 3.4 Hybrid model DSM and “proteins-earliest” model DSM. 95   Figure 3.5 LSU secondary structure and interaction schematic representation of the hybrid model DSM chronology. . 99   Figure 3.6 Half-point distance trend. 104   Figure 3.7 Positions of the PTC and rRNA modules in the LSU. 105       IX   Figure 3.8 Secondary structure of HM. 107   Figure 3.9 Ribbon structure of HM 50S subunit . 108   Figure 3.10 Comparison of L22. . 110   Figure 4.1 Example of the four types of the A-minor interactions. 118   Figure 4.2 A-minor interactions in 16S rRNA 120   Figure 4.3 Interaction networks. . 121   Figure 4.4 Example of contacts comprising SSU r-protein interactions between S14 and S10. . 122   Figure 4.5 Banded DSM model of SSU dependencies from E. coli. 125   Figure 4.6 Secondary structure schematic illustrating chronology of SSU rRNA modules and proteins 128   Figure 4.7 Secondary structures in M. leidyi mt-rRNAs. . 132   Figure 5.1 Intersubunit bridges of the E. coli ribosome. 137   Figure 5.2 DSM chronology of the entire E. coli ribosome 140   Figure 5.3 Adjusted Final Joint chronology . 144   Figure 5.4 Domain mapping graph of the two subunits 146   Figure 6.1 Top view of the 3D ribosomal surface structure using Autodesk Maya. 152   Figure 6.2 Animation frames of insertion steps and chronological milestones. . 156   Figure 6.3 Hydrothermal vent model. . 157   Figure 6.4 Movie capture. . 158   Figure 7.1 Docking trials of r29 and PTC . 165   Figure 7.2 Model of proposed r29-PTC proto-ribosome system. . 166       X   Bibliography Agmon,  I.  (2009).  "The  dimeric  proto-­‐ribosome:  structural  details  and   possible  implications  on  the  origin  of  life."  International  Journal  of   Molecular  Sciences  10(7): 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The boundaries of the 60 rRNA modules in LSU and 29 rRNA modules in SSU are presented here, including ribosomal proteins that are divided into two segments. The number of the first and the last nucleotides is provided. According to the criteria used by Bokov and Steinberg, each rRNA module is considered as an individual double helix or an arrangement of stacked nucleotides according to the A-minor interactions. The boundaries of each module is arranged to make a close position of the 5’ and 3’ termini to each other. All RNA-protein and protein-protein distances were computed using the protein visualization software Rasmol and command line queries, with results captured into text files for further analysis. Supplementary Table 2: Inter-subunit bridges The inter-subunit bridges are listed according to Gao et al (2003). The constituent components of the two subunits from the T. thermophilus ribosome are shown. For each bridge, there exists a counterpart in the E. coli structure. Corresponding numbers in the E. coli are expanded with the description in the study of Gabashvili et al (2000). The locations of each bridge in the two subunits from our models are listed as well.     186   Supplementary Information 3: Manual DSM adjustments The following steps are the record of how manual decisions are overlaid on the DSM process. 1. Transferring the hierarchical model of Bokov and Steinberg into a plausible chronological DSM (PEM). The original LSU DSM dependencies are taken from the work from the Bokov & Steinberg model. Similar criteria are used in the construction of the SSU PEM with the help of a set of Perl script in deciding the rRNA insertion steps and rRNA-protein binding sites. The automated DSM is abandoned as the automated DSM is only upper-triangular matrix and there is not any further algorithm embedded in the software can find the biological boundary path with proteins earliest. 2. LSU PEM to HybridDSM Adjustment               Units   48   45   55   56   53   L22c   Move  Left   After  57   After  48   After  45   After  55   After  56           10   11   37,  31   L32   18,  51   L22e   L3       After  L32   After  51     Move  Right             After  8   modules   After  L22c   After  L22c       After  L22   12   L14     After  22   Reasons    modules    modules    modules    modules    modules   The  first  protein  with  β-­‐strand  pair  structure   Binding  crevice  for  L32   The  second  protein  with  α-­‐helix  structure   Binding  crevice  for  L22e   Composite  of  L22   After  the  basic  a/b  structure  and  binding   crevice  is  formed   After  the  basic  a/b  structure  and  protein-­‐ rRNA  contraint   Units: include rRNA modules and r-proteins 3. Joint Chronology From the assumption adopted in the Hybrid-LSU DSM and BandedSSU DSM models, the same principle of parsimony that the emergence   187     of r-proteins occurs immediately after the forming of the complete protein binding crevice through the rRNA modules’ insertion, but maintaining the order of rRNA events prior to the first LSU proteins. Adjustments   Units     r29,  PTC,   r23     r28,  59,   r22     r25,  58,   r17     r27,  57   Positions   Beginning   Reasons   Three-­‐core  origin   Continual   1st  round  of  rRNA  modules  for  the  three  cores     2nd  round  of  rRNA  modules  for  the  three  cores       Interface   formation  start   Till  interface   formation  end   After  L22e   The  binding  of  the  r29  and  r23  core;  3rd  round  of  rRNA   modules  for  the  two  subunits     8th  round  of  rRNA  modules  for  the  two  subunits  and   the  end  of  band  2  in  BandedDSM   The  first  protein  with  β-­‐strand  structure   The  binding  crevices  for  the  second  protein  with  a-­‐helix   structure;   Interface  between  the  two  subunits,  9  interface   bridges  formed  following  the  auto-­‐sorted  DSM   The  left  units  within  the  3rd  band  of  the  BandedDSM   Continual   The  left  units  within  the  4th  band  of  the  BandedDSM   After  S16     L5,  L18,  L25  appeared   rRNA  modules  within  the  5th  band  of  the  BandedDSM     rRNA  modules  and  r-­‐proteins  of  LSU  following   HybridDSM   Follow  BandedDSM  and  evenly  spread  and  interleaved   in  the  LSU  stages,  with  feedback  from   animation/visualization.       …     r4,  54,  r6         L22c   37,  31,   L32   18 .L22e       r10,  r13,   S7,  S13   r26,  r3,   r2,  S19   5S   r11,  r9,   r12   Units  of   LSU   Units  of   SSU   10   11   12   13   14     Supplementary 4: DVD 1. The movie represents the rRNA modules and r-proteins insertion steps along the ribosomal evolution, following the Joint Chronology DSM result. Format: .m4v Software to open the movie: QuickTime     188   [...]... of nucleic acid gene sequences into proteins in all living creatures It may also be possible that the early ribosome, called the proto -ribosome, was present and influential in the early stages of the RNA world according to the “helicase hypothesis” (Zenkin 2012) that posits that the necessary base pairing of RNA strands in the RNA world required enzymatic separation and that a proto -ribosome may have... into an active protein This section mainly focuses on the translational mechanism of the bacterial ribosomes, which happens in the cell’s cytoplasm Generally, bacterial translation can be divided into three phases, initiation, elongation and termination (Figure 1.3)   Figure 1.3 Overview of the bacterial translation aa-tRNA, aminoacyl-tRNA; EF elongation factor; IF, initiation factor; RF, release factor... Venkatraman Ramakrishnan, Thomas A Steitz and Ada E Yonath for their role in elucidating the crystal structure of the ribosome and its role in the development and understanding of the mechanisms of bacterial ribosome- binding natural product antibiotics Although ribosomes from bacteria, archaea and eukaryotes are responsible for protein synthesis, several significant differences in the structures and... is attached to the new amino acid from the A- site tRNA leaving a deacylated P-site tRNA Following the binding of the GTPase elongation factor G (EF-G), the mRNA shifts by precisely one codon and the tRNAs translocate with respect to the 30S subunit via a rotation of the tRNA molecule from A to P site (Joseph 2003) When an mRNA stop codon moves into the A site, termination occurs The terminal signal... recognized by the class I release factors (RF1 or RF2), which cleaves the nascent polypeptide chain and releases the newly synthesized protein from the ribosome After that, the class II release factors (RF3) triggers the dissociation of class I factors, leaving mRNA and a deacylated tRNA in the P site Next, ribosome recycling factor (RRF) carries out the recycling of ribosome together with EF-G The ribosome. .. proteins across yeast to humans (Venema and Tollervey 1999) Although the core architectures of the prokaryotic and     11   eukaryotic ribosomes are conserved, several additional proteins and new rRNA elements appear in the eukaryotic ribosomes, with important changes in the two subunits Eukaryotic ribosome synthesis largely takes place both in the cell cytoplasm and a specialized nuclear compartment, the. .. clear, such as the first step in initiation, peptidyltransferase reaction, movement of tRNAs and mRNA and so on As the highresolution structures are reported faster using Cryo-EM, an increasing number of functional states structures continues to shed light on the detail of translation of the ribosome involving GTPase factors and other factors (Schmeing and Ramakrishnan 2009) As the core of the ribosome. .. appreciation of the thermal circulation in the element balance of the ocean, these structures further stimulate the advances in the establishment of the hydrothermal-vent origin -of- life theory (Miller and Bada 1988) The discovery of a submarine hydrothermal vent field called Lost City in December 2000 provides one of the most convincing geological sites similar to where life may have originated Although the. .. The interface between the two subunits mainly consists of rRNA The smaller subunit binds to the mRNA through the cleft between the ‘head’ and ‘body’, while the larger subunit binds to the tRNA and the amino acids     10   There are three tRNA binding sites The A site binds to the aminoacyl-tRNA, the P site holds the peptidyl-tRNA with the nascent polypeptide chain, while the deacylated P-site tRNA... (Guerrier-Takada, Gardiner et al 1983) As the discovery of the ribozymes led to speculation that there might be RNA forms capable of self-catalysis at the origin of life, the term ‘RNA World’ was coined by Gilbert on 1986 The premise is accepted that in the early stages of life’s evolution, RNA could cleave, ligate phosphodiester bonds and     16   work as a biosynthetic catalyst and a self-replicating template The . to construct a plausible and detailed evolutional chronology of the 3D structure of the E. coli ribosome, together with a detailed consideration of the environmental factors that may explain. group of passionate people to work with in Hogue’s lab. I could always ask for advice and help. And Kootala Parasuraman Sowmya, our secretary, is always there for us. Also essential to my thesis. animations of the chronology in the Maya software package. Docking supports a potential functional form of the earliest proto -ribosome comprising the PTC and r29, suggesting that the SSU and