No description available.

Few weeks after birth, a switch in peptide elongation factor 1As from EF-1alpha/EF1A-1 to S1/EF1A-2 occurs in brain neurons, heart and skeletal muscles of mammalians. In order to elucidate the reason behind this switch, I studied the expression of both homologous proteins during muscle differentiation and apoptosis and, documented the relation between peptide elongation factors and caspase-3 activation. I found that during in vitro muscle differentiation of L6 myoblasts, a switch in peptide elongation factors 1A occurs as physiologically observed in skeletal muscles. While EF-1alpha/EF1A-1 is expressed in replicating myoblasts, S1/EF1A-2 is solely found in differentiated myotubes where it replaces EF-1alpha/EFIA-1 as the major elongation factor. Similarly, upon serum deprivation-induced apoptosis, a reversion in peptide elongation factors 1A is observed: EF-1alpha/EF1A-1 replaces S1/EF1A-2 and becomes the major form of elongation factor 1A present in dying myotubes. This switch correlates in myotubes with the activation of caspase-3 protein, a cysteine protease involved in apoptosis. When L6 myotubes constitutively express S1/EF1A-2 as caused by adenoviral gene transfer, they become resistant to serum deprivation-induced apoptosis. In contrast, when L6 myotubes are transfected with EF-1alpha/EF1A-1 gene, they die more rapidly from serum deprivation-induced apoptosis than control cells. Transfection using anti-sense EF-1alpha/EF1A-1 gene protects myotubes from apoptotic cell death. Thus, both elongation factor 1As exert opposing effect on muscle survival: while EF-1alpha/EF1A-1 accelerates apoptotic cell death, S1/EF1A-2 protects muscles against apoptosis. / I found that skeletal muscles are the only tissues where, despite the constitutive expression of caspase-3 mRNA, the protein can be absent. Furthermore, I found that while immediately after birth, caspase-3 protein is present in skeletal muscles, a few weeks afterwards, the protein cannot be detected by Western blotting. In skeletal muscle, this change correlates with the observed switch in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 and suggests that caspase-3 is translationally regulated in skeletal muscles. The laboratory previously reported that while EF-1alpha/EF1A-1 protein reappears; S1/EF1A-2 protein becomes absent from regenerating muscles. However, once tissue regeneration is completed, the situation returns to normal as EF-1alpha/EF1A-1 disappears and S1/EF1A-2 reappears to become the only type 1A elongation factor expressed in muscle. / In conclusion, I found that the developmental switch observed in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 partly serves to protect muscle cells from apoptosis. Thus, I am the first to identify a noncanonical function for S1/EF1A-2. (Abstract shortened by UMI.)

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references.

Chu Lai On. / Thesis submitted in: July 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 152-172). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / Table of Content --- p.7 / Abbreviations --- p.12 / Naming system for mutant proteins --- p.13 / Abbreviations for amino acid --- p.14 / Chapter Chapter 1 --- Introduction --- p.15 / Chapter 1.1 --- Structure-function relationship of trichosanthin --- p.18 / Chapter 1.2 --- Properties of acidic ribosomal P-proteins --- p.21 / Chapter 1.3 --- Interaction among P-proteins and trichosanthin --- p.25 / Chapter 1.4 --- Properties of eukaryotic elongation factor 2 and interaction with P-proteins --- p.26 / Chapter 1.5 --- "Objectives and strategy of studying the interaction among trichosanthin, P-proteins and eukaryotic elongation 2" --- p.30 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- General techniques --- p.33 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.33 / Chapter 2.1.2 --- Minipreparation of plasmid DNA using Wizard Plus SV Minipreps DNA purification kit from Promega --- p.34 / Chapter 2.1.3 --- Agarose gel electrophoresis of DNA --- p.36 / Chapter 2.1.4 --- Purification of DNA from agarose gel using Wizard SV Gel and PCR Clean-Up System from Promega --- p.36 / Chapter 2.1.5 --- Polymerase Chain Reaction (PCR) --- p.37 / Chapter 2.1.5.1 --- Basic Protocol --- p.37 / Chapter 2.1.5.2 --- Generation of P2 truncation mutants --- p.38 / Chapter 2.1.5.3 --- Generation of TCS mutants --- p.39 / Chapter 2.1.6 --- Restriction digestion of DNA --- p.41 / Chapter 2.1.7 --- Ligation of DNA fragments --- p.41 / Chapter 2.1.8 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.1.9 --- Staining of protein in polyacrylamide gel --- p.45 / Chapter 2.2 --- Expression and purification of recombinant proteins --- p.46 / Chapter 2.2.1 --- "Bacterial culture, harvesting and lysis" --- p.46 / Chapter 2.2.2 --- Purification of recombinant TCS and mutants --- p.47 / Chapter 2.2.3 --- Purification of acidic ribosomal protein P2 and mutants --- p.48 / Chapter 2.2.4 --- Purification of MBP-fusion proteins --- p.50 / Chapter 2.3 --- Purification of eEF2 from rat livers --- p.51 / Chapter 2.4 --- In vitro binding assay by NHS-activated Sepharose resin --- p.53 / Chapter 2.4.1 --- Coupling of protein sample to NHS-activated Sepharose resin --- p.53 / Chapter 2.4.2 --- In vitro binding of protein sample to coupled NHS-activated resin --- p.54 / Chapter 2.5 --- Ribosome-inactivated activity assay using rabbit reticulocyte lysate in vitro translation system --- p.55 / Chapter 2.6 --- Circular dichroism (CD)spectrometry --- p.57 / Chapter 2.7 --- Isothermal titration calorimetry (ITC) experiment --- p.57 / Chapter 2.8 --- Surface plasmon resonance (SPR) experiment --- p.58 / Chapter 2.8.1 --- Immobilization of P2 onto aminosilane cuvette --- p.58 / Chapter 2.8.2 --- Interaction between eEF2 and immobilized P2 --- p.60 / Chapter 2.9 --- Preparation of Anti-P antibody --- p.61 / Chapter 2.10 --- Western blotting of protein --- p.62 / Chapter 2.11 --- Reagents and buffer --- p.64 / Chapter 2.11.1 --- Reagents for competent cell preparation --- p.64 / Chapter 2.11.2 --- Nucleic acids electrophoresis buffer --- p.65 / Chapter 2.11.3 --- Media for bacterial culture --- p.66 / Chapter 2.11.4 --- Buffers for TCS purification --- p.67 / Chapter 2.11.5 --- Buffers for eEF2 purification --- p.68 / Chapter 2.11.6 --- Reagents for SDS-PAGE --- p.68 / Chapter 2.11.7 --- Reagents and buffers for Western blot --- p.70 / Chapter 2.11.8 --- Reagents and buffers for coupling sample proteins to NHS-activated Sepharose resin --- p.72 / Chapter 2.11.9 --- Reagents and buffers for in vitro binding assay --- p.72 / Chapter 2.11.10 --- Reagents and Buffers for surface plasmon resonance --- p.72 / Chapter 2.12 --- Sequences of primers --- p.73 / Chapter Chapter 3 --- Interaction between TCS and P2 --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Interaction between TCS and P-proteins in rat liver lysate --- p.83 / Chapter 3.3 --- Construction of TCS mutants --- p.85 / Chapter 3.4 --- Expression and purification of TCS mutants --- p.87 / Chapter 3.5 --- Biological assay of TCS mutants --- p.91 / Chapter 3.6 --- Physical interaction of TCS mutants and P2 by surface plasmon resonance (SPR) --- p.94 / Chapter 3.7 --- Discussion --- p.100 / Chapter Chapter 4 --- Mapping the region of P2 that binds TCS and eEF2 --- p.104 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Construction of P2 truncation mutants --- p.106 / Chapter 4.3 --- Expression and purification of P2 truncation mutants --- p.107 / Chapter 4.4 --- Mapping the region of P2 that binds TCS --- p.111 / Chapter 4.4.1 --- Interaction between TCS and P2 mutants by in vitro binding assay --- p.111 / Chapter 4.4.2 --- Interaction study of TCS and P2 mutant by isothermal titration calorimetry (ITC) --- p.116 / Chapter 4.5 --- Mapping the region of P2 that binds eEF2 --- p.120 / Chapter 4.5.1 --- Purification of eEF2 from rat liver --- p.120 / Chapter 4.5.2 --- Physical interaction of P2 and eEF2 by surface plasmon resonance (SPR) --- p.126 / Chapter 4.5.3 --- Interaction between eEF2 and P2 mutants by in vitro binding assay --- p.128 / Chapter 4.6 --- Mapping the C-terminal region of P2 by MBP-fusion proteins --- p.130 / Chapter 4.6.1 --- Construction and purification of MBP-fusion proteins --- p.131 / Chapter 4.6.2 --- "Interaction among eEF2, TCS and MBP-fusion proteins by in vitro binding assay" --- p.133 / Chapter 4.7 --- Discussion --- p.137 / Chapter Chapter 5 --- Effect of C-17 peptide on TCS biological activity --- p.143 / Chapter 5.1 --- Introduction --- p.143 / Chapter 5.2 --- Ribosome-inactivating activity of TCS with C-17 peptide --- p.145 / Chapter 5.3 --- Discussion --- p.147 / Chapter Chapter 6 --- Conclusion and suggestions for future study --- p.149 / References --- p.152 / Appendix --- p.173

Η ΒΥΠ διαθέτει αντίτυπο της διατριβής σε έντυπη μορφή στο βιβλιοστάσιο διδακτορικών διατριβών που βρίσκεται στο ισόγειο του κτιρίου της. / Στα βακτήρια, η μεγάλη ριβοσωματική υπομονάδα αποτελείται από δύο είδη RNA, το 23S και το 5S rRNA, καθώς και 33 πρωτεΐνες. Ο σχηματισμός του πεπτιδικού δεσμού και η απελευθέρωση της πεπτιδικής αλυσίδας επιτελούνται στη μεγάλη υπομονάδα, όπου εδράζεται το καταλυτικό κέντρο της πεπτιδυλοτρανσφεράσης (PTase). Εκτός αυτού, η μεγάλη υπομονάδα περιλαμβάνει το κέντρο προσδέσεως των μεταφραστικών παραγόντων, το οποίο πυροδοτεί τη GTPase δραστηριότητα των G-πρωτεϊνικών παραγόντων, που εμπλέκονται στη μετατόπιση των υποστρωμάτων και άλλες ριβοσωματικές λειτουργίες. Έχει υποτεθεί ότι το 5S rRNA παίζει ουσιώδη ρόλο στη συγκρότηση του κέντρου της PTase και στη μετάδοση σημάτων μεταξύ του καταλυτικού κέντρου και των ριβοσωματικών συστατικών που διεκπεραιώνουν τη μετατόπιση των υποστρωμάτων. Το ιοντικό περιβάλλον φαίνεται να επηρεάζει καθοριστικά τη διαμόρφωση του 5S rRNA. Για παράδειγμα, έχει βρεθεί ότι οι πολυαμίνες δεσμεύονται εκλεκτικά στο 5S rRNA και επηρεάζουν τη δραστικότητα του έναντι του διμεθυλο-θεϊκού, ενός αντιδραστηρίου-ιχνηθέτη της τριτοταγούς δομής του RNA. Αρχικά ελέγξαμε αν υπάρχουν εξειδικευμένες θέσεις πρόσδεσης των πολυαμινών στο 5S rRNA. Στη συνέχεια, με σκοπό να ελέγξουμε αν η πρόσδεση των πολυαμινών επηρεάζει τη λειτουργία του 5S rRNA, 70S ριβοσώματα προγραμματισμένα με πολύ-ουριδυλικό σχηματίσθηκαν από 50S υπομονάδες, ολικά ή εκλεκτικά φωτοσημασμένες με ένα φωτοδραστικό ανάλογο της σπερμίνης και από 30S ακατέργαστες υπομονάδες. Αυτά τα ριβοσώματα είχαν την ικανότητα να δεσμεύουν AcPhe-tRNA ελαφρώς ισχυρότερα, σε σύγκριση με ριβοσώματα συγκροτημένα από φυσικά συστατικά, μη περιέχοντα πολυαμίνες. Το γεγονός αυτό υποδηλώνει ότι η πρόσδεση πολυαμινών στο 5S rRNA επηρεάζει, σε μικρό βαθμό, τη λειτουργία του παράγοντα επιμήκυνσης EF-Tu. Συζευγμένα, όμως, τα εν λόγω ριβοσώματα με tRNAPhe στην Ε-θέση και AcPhe-tRNAστην Ρ-θέση, επέδειξαν ισχυρότερη καταλυτική δραστικότητα και αυξημένη ικανότητα για μετατόπιση των υποστρωμάτων. Τα αποτελέσματα αυτά εισηγούνται σημαντική εμπλοκή των πολυαμινών στο λειτουργικό ρόλο του 5S rRNA κατά την κατάλυση και μετατόπιση των υποστρωμάτων. / In bacteria, the large ribosomal subunit comprises two RNA species, 23S and 5S rRNA, and 33 proteins. Peptide bond formation and peptide release are catalyzed by the large subunit, where the peptidyltransferase (PTase) center is located. In addition to this center, which triggers the GTPase activities of G-protein factors involved in translocation and other ribosomal functions. It has been hypothesized that 5S rRNA plays essential role in assembling the PTase center and mediating signal transmissions between this center and the translocation machinery. Furthermore, the ionic environment seems to affect the conformation of 5S rRNA. For instance, polyamines have been found to bind specifically to 5S rRNA and influence the 5S rRNA reactivity towards dimethyl-sulfate (DMS), a chemical probe of RNA tertiary structure. Initially we examined whether there are specific sites for binding of polyamines. To test whether the binding of polyamines influence the function of 5S rRNA poly(U)-programmed 70S ribosomes were reconstituted from 50S subunits, totally or specifically photolabelled in their 5S rRNA with a photoreactive analogue of spermine, and native 30S subunits. These ribosomes were found to enzymatically bind AcPhe-tRNA better than ribosomes reconstituted from native components. This means, that furnishing 5S rRNA with spermine slightly influences the elongation factor EF-Tu function. However, equipped with tRNAPhe at the A-site and AcPhe-tRNA at the P-site, these ribosomes exhibited higher catalytic activity and enhanced tRNA translocation efficiency. These results suggest an essential impact of polyamines on the functional role of 5S rRNA in catalysis and translocation of translation substrates.