Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.

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.

The bacterial enzyme t-RNA (m1G37) methyltransferase (TrmD) is an ideal anti-microbial drug target since it is found in all eubacteria, serves an essential role during protein synthesis, and shares very little sequence or structural homology with its eukaryotic counterpart, Trm5. TrmD, a homodimeric protein, methylates the G37 nucleotide of tRNA using S-adenosyl-L-methionine (SAM) as the methyl donor and thus enables proper codon-anticodon alignment during translation. The two deeply buried binding sites for SAM seen in X-ray crystallography suggest that significant conformational changes must occur for substrate binding and catalytic turnover. Results from molecular dynamics simulations implicate a flexible loop region and a halo-like loop which may be gating the entrance to the active site. Analysis of simulation trajectories indicates an alternating pattern of active site accessibility between the two SAM binding sites, suggesting a single site mechanism for enzyme activity. Isothermal titration calorimetry (ITC), demonstrates that binding of SAM to TrmD is an exothermic reaction resulting from sequential binding at two sites. A similar mode of binding at higher affinities was observed for the product, S-adenosyl-L-homocysteine (SAH) suggesting that product inhibition may be important in vivo. ITC reveals that tRNA binding is an endothermic reaction in which one tRNA molecule binds to one TrmD dimer. This further supports the hypothesis of a single site mechanism for enzyme function. However, mutational analysis using hybrid mutant proteins suggests that catalytic integrity must be maintained in both active sites for maximum enzymatic efficiency. Mutations impeding flexibility of the halo loop were particularly detrimental to enzyme activity. Noncompetitive inhibition of TrmD was observed in the presence of bis-ANS, an extrinsic fluorescent dye. In silico ligand docking of bis-ANS to TrmD suggests that dye interferes with mobility of the flexible linker above the active site. Because SAM is a ubiquitous cofactor in methyltransferase reactions, analogs of this ligand may not be suitable for drug development. It is therefore important to investigate allosteric modes of inhibition. These experiments have identified key, mobile structural elements in the TrmD enzyme important for activity, and provide a basis for further research in the development of allosteric inhibitors for this enzyme.

As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.

Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

Η ριβονουκλεάση Ρ (RNase P) είναι ένα ριβονουκλεοπρωτεϊνικό ένζυμο, απολύτως απαραίτητο για την βιωσιμότητα του κυττάρου, καθώς είναι υπεύθυνο για την ωρίμανση του 5΄ άκρου των προδρόμων μορίων tRNA. Δραστικότητα RNase P έχει απομονωθεί από όλους τους οργανισμούς που έχουν μελετηθεί μέχρι σήμερα και από τις τρεις φυλογενετικές περιοχές (Βακτήρια, Αρχαία και Ευκαρυώτες), όπως επίσης και από τα ημιαυτόνομα υποκυτταρικά οργανίδια, μιτοχόνδρια και χλωροπλάστες [Frank και Pace 1998, Xiao et al. 2002]. Το ένζυμο αυτό διαθέτει μια RNA υπομονάδα απαραίτητη για την κατάλυση ενώ ο αριθμός των πρωτεϊνών που συμμετέχουν στο ριβονουκλεοπρωτεϊνικό σύμπλοκο ποικίλλει από μια στα βακτήρια έως και δέκα στην RNase P του ανθρώπου [Frank και Pace 1998, Chamberlain et al. 1998, Jarrous 2002]. Η RNA υπομονάδα από τα Βακτήρια και ορισμένα Αρχαία παρουσιάζει καταλυτική δραστικότητα απουσία πρωτεϊνών in vitro, σε υψηλή ιοντική ισχύ [Guerrier-Takada et al. 1983, Pannucci et al. 1999]. Παρότι μέχρι στιγμής καμία τέτοια ιδιότητα δεν έχει εντοπιστεί σε ευκαρυωτική RNA υπομονάδα, πιστεύεται ότι στην πραγματικότητα πρόκειται για ένα ριβοένζυμο [Frank et al. 2000]. Η RNase P από το Dictyostelium discoideum είναι ένα ριβονουκλεοπρωτεϊνικό σύμπλοκο που αποτελείται από RNA και πρωτεϊνικές υπομονάδες οι οποίες είναι απαραίτητες για την δραστικότητα του ολοενζύμου. Η πυκνότητα επιπολής που υπολογίσθηκε για την RNase P από το D. discoideum είναι πολύ χαμηλή σε σχέση με τα χαρακτηρισμένα ολοένζυμα ευκαρυωτικής προέλευσης και είναι παρόμοια με αυτή ενός πρωτεϊνικού μορίου [Stathopoulos et al. 1995]. Παρότι έχει αποδειχθεί ότι το ολοένζυμο αποτελείται από RNA και πρωτεΐνες, πολύ λίγα είναι γνωστά για την ακριβή σύσταση του ριβονουκλεοπρωτεϊνικού συμπλόκου. Πρόσφατα εντοπίστηκε το γονίδιο της RNA υπομονάδας της RNase P από το D. discoideum μέσω συγκριτικής φυλογενετικής ανάλυσης, μήκους 369 νουκλεοτιδίων [Marquez et al. 2005]. Χρησιμοποιώντας τις πρωτεϊνικές υπομονάδες Rpp20 και Rpp40 της RNase P του ανθρώπου πραγματοποιήθηκε αναζήτηση στη τράπεζα δεδομένων της αλληλούχισης του γενωμικού DNA του D. discoideum. Το αποτέλεσμα της αναζήτησης ήταν η εύρεση δύο ανοιχτών πλαισίων ανάγνωσης (drpp20 και drpp40) που κωδικοποιούν δύο πρωτεΐνες (DRpp20 και DRpp40) οι οποίες παρουσιάζουν σημαντική ομολογία με τις υπομονάδες. Η επαγόμενη πρωτεΐνη DRpp20 έχει προβλεπόμενο μοριακό βάρος 26,4 KD, pI 5,6 και επιδεικνύει σημαντική ομοιότητα με την χαρακτηρισμένη πρωτεϊνική υπομονάδα Rpp20 του ανθρώπου (34% ταυτότητα, 56% ομοιότητα σε μήκος 140 αμινοξέων). Όμοια, η πρωτεΐνη DRpp40 έχει προβλεπόμενο μοριακό βάρος 48,2 KD, pI 5,5 και παρουσιάζει σημαντική ομοιότητα με την πρωτεϊνική υπομονάδα Rpp40 (26% ταυτότητα, 45% ομοιότητα σε μήκος 302 αμινοξέων). Παρά την συνολική ομοιότητα, τα μοριακά βάρη των DRpp20 και DRpp40 διαφέρουν σημαντικά σε σχέση με αυτά των ομόλογων πρωτεϊνών τους. Η DRpp20 διαθέτει μια περιοχή χαμηλής πολυπλοκότητας, πλούσια σε κατάλοιπα θρεονίνης, γλουταμίνης και λυσίνης που πιθανόν να συνεισφέρει στο επιπλέον μοριακό βάρος όπως φαίνεται από την στοίχιση με το Clustal W. Τόσο οι επαναλήψεις τρινουκλεοτιδίων γενωμικών περιοχών όσο και οι περιοχές χαμηλής πολυπλοκότητας σε επίπεδο πρωτεΐνης υπάρχουν σε αφθονία στο D. discoideum [Eichinger et al. 2005] και παραμένει να αποδειχτεί εάν αυτά τα χαρακτηριστικά συνεισφέρουν δομικά ή λειτουργικά στις DRpp. Από βιοπληροφορική ανάλυση προκύπτει ότι καμία από τις υπομονάδες των Αρχαίων ή τις εννέα υπομονάδες της ζύμης δεν παρουσιάζει ομοιότητα με τις DRpp20 και DRpp40. Επιπρόσθετα, με την βοήθεια του Pfam αλλά και των προγραμμάτων που συνδέονται με τον MetaServer εντοπίσαμε στην περιοχή 56-126 αμινοξέα της πρωτεΐνης DRpp20 το δομικό μοτίβο των Alba πρωτεϊνών. Η μελέτη των δύο πρωτεϊνών με βάση τον αλγόριθμο PSORT υποδεικνύει ότι και οι δύο πρωτεΐνες έχουν μεγαλύτερη πιθανότητα για χωροθέτηση στον πυρήνα παρά σε οποιοδήποτε άλλο υποκυτταρικό διαμέρισμα. Στην παρούσα εργασία τα υπό μελέτη γονίδια drpp20 και drpp40 κλωνοποιούνται σε φορέα υπερέκφρασης pET-29 και εισάγονται σε δεκτικά κύτταρα BL21(DE3)pLysS. Οι ανασυνδυασμένες πρωτεΐνες απομονώνονται από το κυτταρικό εκχύλισμα με χρωματογραφία συγγενείας σε στήλη νικελίου. Οι πρωτεΐνες DRpp20 και DRpp40 με την μέθοδο που απομονώνονται παραλαμβάνονται σχεδόν στην φυσική τους μορφή όπως προκύπτει και από τα φάσματα του κυκλικού διχρωϊσμού. Οι πρωτεΐνες αυτές χρησιμοποιούνται για την παραγωγή πολυκλωνικών αντισωμάτων καθώς επίσης και για λειτουργικές μελέτες οι οποίες περιγράφονται παρακάτω. Όπως αποδεικνύεται οι πρωτεΐνες DRpp20 και DRpp40 αποτελούν τμήματα του μακρομοριακού συμπλόκου της RNase P. Πολυκλωνικά αντισώματα έναντι των συγκεκριμένων πρωτεϊνών ανιχνεύουν μία ζώνη που συνεκλούεται με την δραστικότητα του ολοενζύμου σε ανάλυση κατά Western. Επιπρόσθετα, η ισχύς αυτής της αλληλεπίδρασης επιτρέπει την κατακρήμνιση καταλυτικά δραστικού ενζύμου με την χρήση των πολυκλωνικών αντισωματών anti-DRpp20 και anti-DRpp40. Μεταξύ των πρωτεϊνών και της RNA υπομονάδας καθώς επίσης του tRNA υποστρώματος αναμένεται να υπάρχουν αλληλεπιδράσεις RNA πρωτεϊνών. Για το λόγο αυτό ελέγχθηκε η ικανότητα των πρωτεϊνών DRpp20 και DRpp40 να αλληλεπιδρούν με μόρια RNA και ιδιαίτερα με μόρια tRNA. Σε μία σειρά πειραμάτων που πραγματοποιήθηκαν δοκιμάστηκαν μόρια tRNA, ολικό RNA αλλά και πλασμιδιακό DNA χωρίς όμως κάποιο αποτέλεσμα στις συνθήκες που πραγματοποιήθηκε η αντίδραση, παρότι άλλες πρωτεΐνες που φέρουν το μοτίβο των Alba πρωτεϊνών έχουν την ικανότητα να αλληλεπιδρούν με μόρια DNA ή δίκλωνα τμήματα RNA. Τέλος, για τις DRpp20, DRpp40 αλλά και το ολοένζυμο, πραγματοποιήθηκε έλεγχος για δραστικότητα ΑΤΡασης κυρίως εξαιτίας της ομολογίας της πρώτης με την Rpp20 του ανθρώπου που διαθέτει τέτοια ιδιότητα, χωρίς να ανιχνεύεται μέσω βιοπληροφορικής ανάλυσης σημαντική ομολογία με αντίστοιχα ένζυμα. Στις συνθήκες που δοκιμάστηκαν δεν ανιχνεύτηκε δραστικότητα ΑΤΡασης που να σχετίζεται με κάποια από τις δύο πρωτεΐνες ή το ολοένζυμο. Ο απώτερος στόχος μας είναι ο προσδιορισμός της ελάχιστης λειτουργικής δομής καθώς και η χαρτογράφηση των αλληλεπιδράσεων πρωτεΐνης-πρωτεΐνης και RNA-πρωτεΐνης στο ολοένζυμο της RNase P. Η ολοκλήρωση της μελέτης θα συμβάλλει στην κατανόηση του καταλυτικού μηχανισμού και της εξέλιξης της ριβονουκλεάσης Ρ από ένα αρχέγονο ριβοένζυμο σε ένα υψηλά οργανωμένο ριβονουκλεοπρωτεϊνικό σύμπλοκο. / Ribonuclease P (RNase P) is a ubiquitous and essential ribonucleoprotein enzyme that matures the 5´ end of all primary tRNA transcripts. It has been studied from a variety of organisms, representing the three domains of life (Bacteria, Archaea and Eukarya), as well as from the major subcellular organelles, mitochondria and chloroplasts [Frank and Pace 1998, Xiao et al. 2002]. RNase P enzymes contain a similar in size RNA subunit which is absolutely required for catalysis. However, the size and number of protein subunits of the holoenzyme varies significantly, from one small subunit in bacteria to ten subunits in human RNase P [Frank and Pace 1998, Chamberlain et al. 1998, Jarrous 2002]. The RNA subunit from bacteria and some archaea is catalytically active in vitro in high ionic strength and in the absence of the protein fraction of RNase P [Guerrier-Takada et al.1983, Pannucci et al. 1999]. No such activity has been proven yet for eukaryotic RNA subunit but is still considered to be intrinsically a ribozyme [Frank et al. 2000]. Dictyostelium discoideum RNase P holoenzyme is a ribonucleoprotein complex, consisted of RNA and proteins essential for catalytic activity. Considering its buoyant density, D. discoideum RNase P exhibits one of the most proteinaceous idiosyncrasies, among the characterized holoenzymes of eukaryotic origin [Stathopoulos et al. 1995]. Although it has been established that this enzyme contains both RNA and protein components, very little is known on the exact composition of the ribonucleoprotein complex. A recent report identified a putative RNA subunit of D. discoideum RNase P of length of 369 nucleotides through phylogenetic comparative analysis [Marquez et al. 2005]. Genomic analysis of the available data from D. discoideum sequencing projects, revealed among others the existence of two open reading frames (drpp20 and drpp40) encoding two proteins (DRpp20 and DRpp40) that show significant similarity to previously characterized proteins subunits Rpp20 and Rpp40 from human RNase P. The encoded protein DRpp20 has a predicted molecular mass of 26,4 KD, pI 5,6 and exhibits significant similarity to characterized human RNase P protein subunit, Rpp20 (34% identity, 56% similarity at a length of 140 amino acids). Likewise, the protein DRpp40 of a predicted mass of 48,2 KD and pI 5,5, displays significant similarity to its human counterpart, Rpp40 (26% identity, 45% similarity at a length of 302 amino acids). DRpp20 harbors a region of low complexity (rich in threonine residues) which confers to higher MW in comparison with the human homologue. Such regions have not been encountered so far in proteins of this kind in other organisms. Tandem repeats at the genomic and the protein level, are abundant in D. discoideum [Eichinger et al. 2005] and it remains to be proven if these features contribute to the structure and function of DRpp proteins. To the best of our knowledge no homologues of DRpp20 and DRpp40 have been identified in yeast and archaeal RNase P enzymes. Additionally, pattern search of the D. discoideum protein sequences using MetaServer and Pfam prediction tools identified a DRpp20 region (amino acids 56 to 126) that bears similarity to the Alba domain. PSORT analysis of DRpp20 and DRpp40 predicts that these proteins are likely to localise into the nucleus. In this study the putative ORFs were subcloned into pET-29 expression vector and the recombinant vectors were used for the transformation of BL21(DE3)pLysS. The recombinant polypeptides were purified from the cell extract using Ni2+-nitriloacetic acid agarose column. The purified proteins are isolated in their native form as supported by circular dichroism analysis of the preparations. These preparations were used for the production of polyclonal antibodies as well as functional studies as described below. DRpp20 and DRpp40 are functionally associated with the RNase P ribonucleoprotein catalytic complex. Using anti-DRpp20 and anti-DRpp40 polyclonal antibodies we ascertained the concurrence of DRpp20 and DRpp40 with purified RNase P activity after standard purification schemes. Moreover, the nature of this association permits the precipitation of RNase P activity through antigen-antibody interaction using the same antibodies. RNA-proteins interactions between the protein subunits, the RNA moiety and/or the RNA substrate are expected in the holoenzyme complex, and therefore the ability of DRpp40 and DRpp20 to bind to RNA molecules was investigated. In a series of experiments using a variety of binding partners (plasmids, tRNAs and total RNA), we did not detect any DNA or RNA binding properties for DRpp20 and DRpp40, although other proteins that contain the Alba core interact with DNA or double stranded RNA regions. Although neither DRpp20 nor DRpp40 harbours an ATPase domain, we tested DRpp40 and DRpp20 for ATPase activity mostly due to the latter homology with human Rpp20, which was shown to have ATPase activity. We could not detect any ATPase activity associated with aforementioned proteins or holoenzyme. Our future prospects are the determination of minimal catalytic core and the complete mapping of all protein-protein and RNA-protein interactions within RNase P holoenzyme. The completion of this project will contribute in a decisive manner to the understanding of both the catalytic mechanism and the evolution of RNase P from a primordial ribozyme to a highly organized ribonucleoprotein complex.

Η ριβονουκλεάση P είναι το ένζυμο το οποίο αναλαμβάνει την δημιουργία του 5´ ώριμου άκρου όλων των πρόδρομων μορίων tRNA. Πρόκειται για ένα ριβονουκλεο-πρωτεϊνικό σύμπλοκο το οποίο εντοπίζεται στα κύτταρα των οργανισμών και από τις τρεις κύριες φυλογενετικές περιοχές, τα Βακτήρια, τα Αρχαία και τους Ευκαρυώτες. Αποτελείται από μια υπομονάδα RNA απαραίτητη για την κατάλυση, ενώ το μέγεθος και ο αριθμός των πρωτεϊνικών υπομονάδων ποικίλλει από μια μικρή στα βακτήρια έως δέκα πρωτεΐνες στο ολοένζυμο που απομονώνεται από τα ανθρώπινα κύτταρα. Η υπομονάδες RNA των βακτηρίων καθώς επίσης και μερικών αρχαίων μπορούν να καταλύσουν την αντίδραση ωρίμανσης του tRNA απουσία της πρωτεΐνης in vitro, είναι δηλαδή ριβοένζυμα. Η ανακάλυψη αυτή διεύρυνε τις αντιλήψεις μας για τις ιδιότητες των βιομορίων και επανέφερε στο προσκήνιο την θεωρία του κόσμου του RNA. Στο ευκαρυωτικό ριβοένζυμο, ο ρόλος των πρωτεϊνών είναι πιο ουσιαστικός, καθώς η υπομονάδα RNA φαίνεται ότι χάνει μεγάλο μέρος της λειτουργικής της ανεξαρτησίας. Η διαλεύκανση των λειτουργών της κάθε υπομονάδας θα δώσει σημαντικές πληροφορίες για την εξέλιξη της RNase P από ένα αρχέγονο ένζυμο σε ένα πολύπλοκο ριβονουκλεοπρωτεϊνικό σύμπλοκο. Η RNase P από το Dictyostelium discoideum διαθέτει μια απαραίτητη για την δραστικότητα υπομονάδα RNA όπως και όλα τα ένζυμα αυτού του είδους. Παράλληλα διαθέτει έντονο πρωτεϊνικό χαρακτήρα καθώς διαθέτει την χαμηλότερη πυκνότητα επιπολής σε σχέση με ένζυμα RNase P από άλλους οργανισμούς. Οι πληροφορίες αυτές προέρχονται από τον αρχικό χαρακτηρισμό του ενζυμικού συμπλόκου, και δεν παρέχουν στοιχεία για την ακριβή σύστασή του. Στην παρούσα μελέτη, πραγματοποιήθηκε κλωνοποίηση και χαρακτηρισμός ενός από τα γονίδια που εντοπίστηκαν στο γονιδίωμα του Dictyostelium, ομόλογα προς χαρακτηρισμένα γονίδια από τον άνθρωπο και άλλους ευκαρυώτες. Το γονίδιο drpp30 κωδικεύει μια πρωτεΐνη 40.7 kDa, σημαντικά μεγαλύτερη από τις ομόλογες της. Η πρωτεΐνη DRpp30 υπερεκφράστηκε σε βακτηριακά κύτταρα, και μετά τον χρωματογραφικό καθαρισμό της χρησιμοποιήθηκε για την παρασκευή πολυκλωνικών αντισωμάτων. Η συμμετοχή της DRpp30 στο μακρομοριακό σύμπλοκο της RNase P πιστοποιήθηκε με ανοσοβιοχημική προσέγγιση, ενώ η ανασυνδυασμένη πρωτεΐνη προσδένει τo pre-tRNA υπόστρωμα του ενζύμου, καθώς και την υπομονάδα RNA in vitrο. Το μοντέλο ομολογίας της DRpp30 βάσει της κρυσταλλικής δομής της ορθόλογης Ph1877 από τα αρχαία, φανερώνει ότι η πρωτεΐνη αποκτά τη δομή αβ βαρελιού (ΤΙΜ barrel fold). Κατά τη διάρκεια της διατριβής, οι προσπάθειες για τον εντοπισμό του γονιδίου της RNA υπομονάδας ήταν σε εξέλιξη, όταν το εν λόγω γονίδιο αναγνωρίστηκε μέσω φυλογενετικών συγκρίσεων από την ομάδα του Norman Pace. Το μετάγραφο του γονιδίου εντοπίστηκε σε ενεργά κλάσματα RNase P, και παράλληλα εντοπίστηκε και ένα μικρότερο μετάγραφο του ίδιου γονιδίου. Προσδιορίστηκαν τα ακριβή 5´και 3´ άκρα των δύο αυτών μορίων και ακολούθησε κλωνοποίηση τους. Τα in vitro μετάγραφα των δύο κλωνοποιημένων αλληλουχιών μπορούν να υποκαθιστούν την ενδογενή RNA υπομονάδα του ολοενζύμου in vitro, ενώ δεν εντοπίστηκε έως τώρα ενζυμική δραστικότητα που να σχετίζεται με τα δύο αυτά μόρια. / Ribonuclease P is a ubiquitus ribonucleoprotein enzyme, responsible for the production of the 5´ mature ends of all precursor tRNA molecules. RNase P endonucleolytic activity has been isolated from organisms representing the three domains of life, namely Bacteria, Archaea and Eukarya. It has been shown to contain an essential RNA subunit and one (Bacteria) or more (Archaea, Eukaryotes) proteins. The RNase P RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea have lost most of their functionality and require protein subunits for activity. RNase P has been characterized biochemically and genetically in several systems, and structures for both RNA and protein subunits have emerged. The integration of structural and functional data is slowly forming a scenario for the evolution of RNase P from an ancient enzyme to a highly organized ribonucleoprotein complex. Dictyostelium discoideum RNase P harbors an essential RNA subunit, and has high protein content, as judged by its low boyant density. Nevertheless, our knowledge on the exact composition was limited. In the current study, a gene showing significant similarity to human Rpp30 RNase P protein subunit was identified in Dictyostelium genome. The gene encodes a protein (DRpp30) which is significantly larger than its homologues, due to an unusual C-terminus. The gene was cloned, overexpressed, and was used for the production of polyclonal antibodies. The participation of DRpp30 in the macromolecular complex of RNase P was verified by an immunobiochemical approach. The recombinant protein was shown to bind specifically both the RNase P RNA subunit and the pre-tRNA substrate in vitro, thus giving a first insight of its role in the holoenzyme complex. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold. While our efforts to isolate the gene encoding the RNA subunit of D. discoideum RNase P were in progress, Norman Pace and his group identified it through phylogenetic comparison. The full transcript of the gene was detected in active RNase P samples along with a smaller transcript of the same gene. The exact 5´and 3´ ends of both transcripts were identified and were cloned. Both these transcripts can substitute the endogenous RNA subunit in vitro, but no enzymatic activity associated with these RNA molecules could be detected so far.

The repertoire of cellular functions mediated by Ribonucleic acid (RNA) molecules have expanded considerably during the last two decades. The role played by RNA in controlling and regulating gene expression in viruses, prokaryotes and eukaryotes has been a matter of continuous investigations. This interest has arisen primarily due to the discoveries of cisacting RNA structures like riboswitches, ribosensors and frameshift elements, which are found in either the 5’-, 3’-untranslated regions of mRNA or in the open reading frames. These structures control gene expression at the level of translation by either sequestering the Shine-Dalgarno (SD) sequence to regulate translation initiation or modulating ribosomal positions during an active translation process. Very often, these structures comprise of an RNA pseudoknot and it has been observed that these pseudoknots exist in a dynamic equilibrium with other intermediate structures. This equilibrium could be shifted by several factors including presence of ions, metabolites, temperature and external force. RNA pseudoknots represent the most versatile and ubiquitous class of RNA structures in the cell, whose unique folding topology could be exploited in a number of ways by the cellular machinery. In this thesis, a thorough study of programmed -1 ribosomal frameshifting (-1 PRF) process, which is a well known gene regulation event employed by many RNA viruses, was carried out. -1 PRF is a translation recoding process, necessary for viruses to main-tain a stoichiometric ratio of structural: enzymatic proteins. This ratio varies among different viral species. At the heart of this process, lies an RNA pseudoknot accompanied by a seven nucleotide long sequence motif, which pauses an actively translating ribosome on mRNA and causes it to shift its reading frame. The frameshift inducing efficiency of pseudoknot depends on multiple factors, for example the time scale of ribosomal pause and RNA unfolding, subsequent refolding of structure to native/intermediate states and/or environment conditions. With the aim of illustrating the fundamentals of the process, multiple factors involved in -1 PRF were studied. Chapters 2-4 represent distinct aspects of -1 PRF process, while Chapter 5 discusses a different work concerned with nucleocytoplasmic transport of tRNA carried out by nuclear export receptor Exporting. Chapter 1 gives an overview of the different regulatory activities with which RNA structures and sequences are found to be associated and the evolution of these stud-ies. It discusses the different types of structural motifs found to constitute tertiary RNA structure and secondary structure prediction and determination techniques. A brief description of ab initio RNA structure modeling and other relevant tools and methodologies used in this work has been presented. Details of techniques used in each study have been provided in relevant chapters. Chapter 2 describes how local factors like ionic conditions, hydration patterns, presence of protonated residues and single residue mutations affect the structural dynamics of an RNA pseudoknot involved in -1 PRF from a plant luteovirus. Single residue mutations in the loop regions or certain base-pair inversions in the stem regions of pseudoknot increase the frameshift inducing ability of the pseudoknot structure, while some others decrease this efficiency. However, it was not clear how the changes made to the wild-type (WT) RNA pseudoknot from Beet Western Yellow Mosaic virus were affecting the global structure in terms of its dynamics and other parameters. To study this, multiple all-atom molecular dynamics simulations (MD) were performed on WT and mutant structures created in silico. The effect of presence and absence of magnesium ions on the structural geometry was also studied. The analysis was done to identify the increase/decrease in the number of hydrogen bonds formed by Watson-Crick base-pairs in stem region or non Watson-Crick pairs between stem and loop. Ionic and water densities were analyzed and the role of potential ribosome-pseudoknot interaction was elaborated. With the aim of mimicking ribosome induced unfolding of an RNA pseudoknot, steered molecular dynamics pulling experiments were performed. This work was done primarily to understand the unfolding pathway of Hairpin(H)-type pseudoknots in general and the intermediate structures formed. Chapter 3 describes the thermodynamics and mechanics associated with the mechanical pulling of -1 PRF inducing RNA pseudoknot and its mutants described in previous chapter. Analysis of the trajectories reveal relative unfolding patterns in terms of disruption of various hydrogen bonds. This study allowed us to pinpoint the kind of intermediate structures being formed during pulling and whether these intermediate structures correspond to any known secondary structures, such as simple stem-loops. This information could be used for gaining insights into the folding pathways of these structures. An RNA pseudoknot stimulates -1 PRF in conjunction with a heptanucleotide “slippery site” and an intervening spacer sequence. A comprehensive study of analyzing the sequence signatures and composition of all overlapping gene segments harboring these frameshift elements from four different RNA virus families was carried out. Chapter 4 describes the sequence composition of all overlapping gene segments in Astroviridae, Coronaviridae, Retroviridae and Luteoviridae viral families which are known to employ -1 PRF process for maintaining their protein products. Sequence analysis revealed preference for GC bases in the structure forming sequence regions. A comparative study between multiple sequence alignment and secondary structure prediction revealed that while pseudoknots have a clear preference for specific base-pairs in their stem regions, viral families that employ a hairpin loop as -1 PRF structure, doesn’t show this preference. Information derived from secondary structure prediction was then used for RNA ab initio modeling to generate tertiary structures. Furthermore, the structural parameters were calculated for the helices of the frameshift inducing pseudoknots and were compared with the values calculated for a set of non -1 PRF inducing H-type pseudo-knots. This study highlighted the differences between -1 PRF pseudoknots and other H-type pseudoknot structures as well as specific sequence and structural preferences of the former. Chapter 5 discusses the dynamics of a tRNA transport factor Exportint (Xpot), which transports mature tRNA molecules from nucleus to cytoplasm and belongs to Importitβ family of proteins. The global conformational dynamics of other transport receptors has been reported earlier, using coarse-grained modeling and Elastic Network Models (ENMs), but a detailed description of the dynamics at an all-atomic resolution was lacking. This transport requires association of Xpot with RanGTP, a G-protein, in the nucleus and hydrolysis of RanGTP in the cytoplasm. The chain of events leading to tRNA release from Xpot after RanGTP hydrolysis was not studied previously. With these objectives, several molecular complexes containing Xpot bound to Ran or tRNA or both in the GTP and GDP ligand states as well as free Xpot structures in nuclear and cytosolic forms were studied. A combination of conventional and accelerated molecular dynamics simulations was used to study these molecular complexes. The study highlighted various aspects associated with tRNA release and conformational change which occurs in Xpot in cytosolic form. The nuclear to cytosolic state transition in Xpot could be attributed to large fluctuations in C-terminal region and dynamic hinge-points located between specific HEAT repeats. A secondary role of Xpot in controlling the quality of tRNA transport has been proposed based on multiple sequence and structure alignment with Importin-β protein. The loss of critical contacts like hydrogen bonds and salt bridges between Xpot/Ran and Xpot/tRNA interface was evaluated in order to study the initial effects of RanGTP hydrolysis and how it influences receptor-cargo binding. This study revealed various aspects of tRNA transport process by Xpot, not understood previously. The results presented in this thesis illustrate the role of RNA sequence elements and pseudoknots present in RNA viruses in modulating -1 PRF process and how multiple environmental factors affect -1 PRF inducing ability of the structure. From the studies of Xpot and its complexes, the effects of GTP hydrolysis leading to tRNA dissociation have been presented and the progression of conformational transition in Xpot after tRNA dissociation has been highlighted. Chapter 6 summarizes major conclusions of this thesis work. The refolding of single stranded RNA chains, subjected to a previous unfolding simulation is studied. Appendix A describes this work and initial results. Appendix B describes the effect of improved molecular dynamics force fields, containing corrections for χ torsion angle for RNA, on the conformation of tertiary RNA structures. Part of the work presented in this thesis has been reported in the following publications. 1.Asmita Gupta and Manju Bansal. Local Structural and Environmental Factors De-fine the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process. J. Phys. Chem. B. 118 (41), pp 11905-11920. 2014 2.Asmita Gupta, Senthilkumar Kailasam and Manju Bansal. Insights Into Nucleo-cytoplasmic Transport of tRNA by Exportin-t. Manuscript under review. List of manuscripts that are being prepared from the work reported in Chapter 3 in this thesis. 1 Asmita Gupta and Manju Bansal. The role of sequence effects on altering the un-folding pathway of an RNA pseudoknot: a steered molecular dynamics study. Manuscript in preparation. 2 Asmita Gupta and Manju Bansal. Molecular basis for nucleocytoplasmic transport of tRNA by Exportin-t. Journal of Biomolecular Structure and Dynamics, May;33 Suppl 1:59-60, 2015

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.

As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.