Το ριβόσωμα παρέχει την πλατφόρμα πάνω στη οποία το mRNA αναγνωρίζεται και αποκωδικοποιείται από τα μεταφορικά RNAs. Δρα καταλυτικά ως ριβοένζυμο και συνθέτει την αντίστοιχη αλληλουχία αμινοξέων. Σημαντικά λειτουργικό κομμάτι του ριβοσώματος αποτελεί το τούνελ εξόδου, το οποίο βρίσκεται στη μεγάλη υπομονάδα του ριβοσώματος και αποτελεί το κανάλι, μέσω του οποίου οι νεοσυντιθέμενες πεπτιδικές αλυσίδες εξέρχονται στο κυτταροδιάλυμα. Νεότερες ερμηνείες προσδίδουν στο τούνελ εξόδου έναν πιο ενεργό ρόλο, καταδεικνύοντας τη σπουδαιότητά του όχι μόνο ως διάδρομος εξόδου της πεπτιδικής αλυσίδας, αλλά και ως λειτουργική περιοχή του ριβοσώματος με σημαντικό ρόλο στη ρύθμιση της πρωτεϊνικής σύνθεσης. Το δίκτυο επικοινωνίας μεταξύ τούνελ και πεπτιδυλοτρανσφεράσης δεν έχει ακόμα χαρτογραφηθεί και μελετηθεί πλήρως, υπάρχουν όμως μέχρι στιγμής πολυπληθείς αναφορές που επιβεβαιώνουν ότι μέρος του αποτελούν κυρίως βάσεις του 23S rRNA, συστατικά του τοιχώματος της εισόδου στο τούνελ, και βάσεις ευρισκόμενες στο κέντρο της πεπτιδυλοτρανσφεράσης, συντηρημένες εξελεικτικά, γνωστές από την αλληλεπιδρασή τους με αναστολείς της πεπτιδυλοτρανσφεράσης και κυρίως με αναστολείς της οικογένειας των μακρολιδίων. Στην παρούσα διατριβή μελετήσαμε την αλληλεπίδραση των χαρακτηριστικών αυτών βάσεων που συγκροτούν το δίκτυο επικοινωνίας μεταξύ του τούνελ εξόδου και της πεπτιδυλοτρανσφεράσης με τη βοήθεια νέων μακρολιδίων τρίτης γενιάς, των κετολιδίων, που φέρουν επί πλέον και άτομα φθορίου, παρέχοντας έτσι στο μόριο μεγαλύτερη συγγένεια για φορτισμένες ομάδες. Σύμφωνα με τα αποτελέσματά μας, αποτελούν εξαιρετικά υποσχόμενους αντιμικροβιακούς θεραπευτικούς παράγοντες, και αναστέλλουν ισχυρά την μετάφραση. Παράλληλα καταλαμβάνουν αμοιβαία αποκλειόμενες θέσεις στο ριβόσωμα, στην είσοδο του τούνελ, αλληλεπιδρώντας με τις βάσεις Α2058, Α2059, Α2062 και Α752. Επειδή όλες οι παραπάνω βάσεις αποτελούν μέρος του δικτύου επικοινωνίας τούνελ-πεπτιδυλοτρανσφεράσης, θα μπορούσαμε να συμπληρώσουμε τον ήδη υπάρχοντα μηχανισμό δράσης των μακρολιδίων (drop-off, λόγω αύξησης της koff) με την επικοινωνία του τούνελ με την πεπτιδυλοτρανσφεράση, ώστε να απενεργοποιηθεί η τελευταία με τελική κατάληξη τη διακοπή της πρωτεϊνικής σύνθεσης και την επαγωγή του drop-off. Για την περαιτέρω μελέτη της αλληλεπίδρασης του τούνελ με την νεοσυντιθέμενη πεπτιδική αλυσίδα, σχεδιάσθηκαν νέα πεπτιδυλο παράγωγα της χλωραμφανικόλης, τα οποία προσομοιάζουν πεπτιδυλο-tRNAs προσδεδεμένα στην Α-θέση του ριβοσώματος, με την ολιγοπεπτιδική αλληλουχία να εκτείνεται βαθύτερα στο τούνελ, υιοθετώντας μία ανοικτή διαμόρφωση. Όπως διαπιστώσαμε με συναγωνιστικές μελέτες με ραδιενεργό ερυθρομυκίνη, τα συγκεκριμένα ανάλογα καταλαμβάνουν την Α-θέση στο κέντρο της πεπτιδυλοτρανσφεράσης, ενώ η συγγένεια του κάθε αναλόγου ως αναστολέα της λειτουργίας του ριβοσώματος, είναι απόλυτα ιδιοσυγκρατική, καθώς εξαρτάται από την αλληλουχία των αμινοξέων που συγκροτούν το πεπτίδιο. Μελετώντας το αποτύπωμα των ενώσεων αυτών στο ριβόσωμα, διαπιστώσαμε ότι ενώ η βάση της χλωραμφαινικόλης διατηρεί τη θέση της στη περιοχή Α, το πεπτίδιο εισέρχεται βαθειά στην είσοδο του τούνελ, αλληλεπιδρώντας με την βάση Α752. Συνεπώς τα πεπτιδυλο ανάλογα της χλωραμφαινικόλης, τα οποία συμπεριφέρονται ως ισχυροί αναστολείς της μετάφρασης, αποτελούν κατάλληλα εργαλεία μελέτης της αλληλεπίδρασης μεταξύ της νεοσυντιθέμενης πεπτιδικής αλληλουχίας και στοιχείων του τούνελ εξόδου του ριβοσώματος. / Ribosomes translate the genetic code into proteins in all living cells with extremely high efficiency, owing to their inherent flexibility and to their spectacular architecture. These large ribonucleoprotein particles synthesize proteins in all cells, using messenger RNA as the template, confirming that the ribosome is indeed a ribozyme, and aminoacyl-transfer RNAs as substrates. As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. New studies indicate that in some cases, the tunnel plays a more active role. Accumulated evidence had definitely concluded that ribosomal tunnel is not only a passive conduit for the nascent chains but a rather functionally important compartment, where specific peptide sequences establish direct interactions with the tunnel, talking back to the ribosome in order to regulate peptide synthesis, leading to translational stalling. In this study, we have investigated functional interactions between distinct locations of the ribosomal tunnel and specific residues of the peptidyltransferase, using new macrolides, known as ketolides, which carry also fluorine attached to the lactone ring either directly or indirectly. According to our results, the new antibiotics exhibited better antimicrobial activity, inhibiting strongly the translational apparatus and could be useful tools in the future for treatment of bacterial infections. Binding studies with radiolabeled erythromycin revealed that these drugs bound competitively with erythromycin at the large ribosomal subunit, with extremely low dissociation constants. In parallel, RNA footprinting indicated that the new ketolides occupy the main macrolide binding site in the ribosome, which is located at the entrance of the exit tunnel adjacent to the peptidyltransferase center. These drugs interact with A2058, A2059 and A2062, as well as their extended heteroaromatic alkyl-aryl side chain penetrates deeper in the tunnel protecting A752 of Helix 35, interacting with basepair A752-U2609. To explore further this interaction, we have synthesized new peptidyl conjugates of chloramphenicol, which resemble nascent peptidyl-tRNA chains bound to the A-site of the ribosome, with their peptide sequence located deeper within tunnel, adopting an extended configuration. According to our data, these compounds did indeed bind to the peptidyl transferase center, competing with radiolabelled- chloramphenicol for binding to the ribosome. The binding of each analog, as well as its inhibitory activity of ribosomal function, is absolutely idiosyncratic, depending on the sequence of amino acid of peptide moiety. Studying the footprinting pattern of these compounds on the ribosome, we confirmed that while the chloramphenicol base maintains its position on the A-site, the peptide moiety penetrates deeper in the entrance of the exit tunnel, interacting with nucleotide A752. Therefore, we believe that these chloramphenicol peptides could be useful tools for probing nascent polypeptide chain interaction with the ribosome.

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.

Protein synthesis is a process by which organisms manufacture their proteins that perform various cellular activities either alone or in combination with other similar or different molecules. In eubacteria, protein synthesis proceeds at a rate of around 15 amino acids per second. The ribosomes, charged tRNAs and mRNAs can be considered as the core components of protein synthesis system which, in addition, involves a panel of non-ribosomal proteins that regulate the speed, specificity and accuracy of the process. Peptidyl-tRNA hydrolase (Pth) and ribosome recycling factor (RRF) are two such non-ribosomal proteins involved in protein synthesis. These two proteins are essential for eubacterial survival and the work reported in this thesis involves structural characterization of these two proteins from the bacterial pathogen, Mycobacterium tuberculosis. The protein structures were solved using established techniques of protein crystallography. Hanging drop vapour diffusion method and crystallization under oil using microbatch plates were the methods employed for protein crystallization. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator in all the cases. The data were processed using DENZO and MOSFLM. The structures were solved by molecular replacement method using the program PHASER. Structure refinements were carried out using programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, CHIMERA, and PYMOL were used for structure validation and analysis of the refined structures. Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA that has dropped off from ribosome before reaching the stop codon, in order to avoid the toxicity resulting from peptidyl-tRNA accumulation and to free the tRNA to make it available for further rounds in protein synthesis. To begin with, the structure of the enzyme from M. tuberculosis (MtPth) was determined in three crystal forms. This structure and the structure of the same enzyme from Escherichia coli (EcPth) in its crystal differ substantially on account of the binding of the C-terminus of the E.coli enzyme to the peptide binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in open conformation when the enzyme is in the free state as in the crystals of MtPth. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the EcPth crystal mimics the peptide-bound conformation of the enzyme. The peptide stretch involving a loop and a helix, referred to earlier, now closes on the bound peptide. Concurrently, a gate connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of the two residues. Thus, the crystal structure of MtPth when compared with that of EcPth, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. A discrepancy between the X-ray results and NMR results, which subsequently became available, led to X-ray studies on new crystal forms of the enzyme. The results of these studies and those of the enzyme from different sources that became available, confirmed the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding site and tRNA binding site. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movement is not, however, observed in the NMR structure of MtPth. The discrepancy between the X-ray and NMR structures of MtPth in relation to the functionally important plasticity of the molecule, referred to earlier, also led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures are available appears to confirm this correlation. In consonance with the reported results of the investigation in cellular components and aqueous solutions, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important for function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule. After termination of protein synthesis at the stop codon, the ribosome remains as a post-termination complex (PoTC), consisting of the 30S and the 50S subunits, mRNA and a deacylated tRNA. This complex has to be disassembled so that the ribosome is available for the next round of translation initiation. Ribosome recycling factor (RRF) binds to ribosome and in concert with elongation factor G (EF.G), performs the recycling of ribosome that results in disassembly of PoTC. The structure of this L-shaped protein with two domains connected by a hinge, from Mycobacterium tuberculosis (MtRRF) was solved previously in our laboratory. The relative movement of domains lies at the heart of RRF function. Three salt bridges were hypothesized to reduce the flexibility of MtRRF when compared to the protein from E.coli (EcRRF), which has only one such salt bridge. Out of these three bridges, two are between domain 1 and domain 2, whereas the third is between the hinge region and the C-terminus of the molecule. These salt bridges were disrupted with appropriate mutations and the structure and activity of the mutants and their ability to complement EcRRF were explored. An inactive C-terminal deletion mutant of MtRRF was also studied. Major, but different, structural changes were observed in the C-terminal deletion mutant and the mutant involving the hinge region. Unlike the wild type protein and the other mutants, the hinge mutant complements EcRRF. This appears to result from the increased mobility of the domains in the mutant, as evidenced by the results of librational analysis. In addition to the work on PTH and RRF, the author was involved during the period of studentship in carrying out X-ray studies of crystalline complexes involving amino acids and carboxylic acids, which is described in the Appendix of the thesis. The complexes studied are that of tartaric acid with arginine and lysine.