Methods are being developed to produce “designer proteins” from unnatural amino acids that are added into specific locations by the ribosome using an altered mRNA. To date, over seventy unnatural amino acids have been incorporated at specific sites in proteins by in vitro biosynthetic methods using chemically acylated-tRNAs and in vivo protein mutagenesis based on orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Lanthanum-mediated aminoacylation of cis-diols provides a general and selective method for the one-step preparation of aminoacyl-tRNA. The nature of this biomimetic process was studied for the reaction of ribonucleosides and nucleotides with N-t-Boc-protected aminoacyl ethyl phosphates. Successful aminoacylation was also achieved with unprotected aminoacyl ethyl phosphates. This method was extended for the aminoacylation of tRNA and analyzed by reversed-phased HPLC and MALDI-MS. These results will provide an insight to the ultimate goal of lanthanum-mediated direct acylation of tRNA and its applications in in vitro site-specific incorporation of unnatural amino acids.

Methods are being developed to produce “designer proteins” from unnatural amino acids that are added into specific locations by the ribosome using an altered mRNA. To date, over seventy unnatural amino acids have been incorporated at specific sites in proteins by in vitro biosynthetic methods using chemically acylated-tRNAs and in vivo protein mutagenesis based on orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Lanthanum-mediated aminoacylation of cis-diols provides a general and selective method for the one-step preparation of aminoacyl-tRNA. The nature of this biomimetic process was studied for the reaction of ribonucleosides and nucleotides with N-t-Boc-protected aminoacyl ethyl phosphates. Successful aminoacylation was also achieved with unprotected aminoacyl ethyl phosphates. This method was extended for the aminoacylation of tRNA and analyzed by reversed-phased HPLC and MALDI-MS. These results will provide an insight to the ultimate goal of lanthanum-mediated direct acylation of tRNA and its applications in in vitro site-specific incorporation of unnatural amino acids.

Thesis (Ph. D.) -- California Institute of Technology, 2009. / Advisor and committee chair names found in the thesis' metadata record in the digital repository. Title from home page (viewed 06/21/2010). Includes bibliographical references.

AN ABSTRACT OF THE DISSERTATION OF MANISHA DEOGHARIA, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry presented on May 16, 2018, at Southern Illinois University, Carbondale TITLE: PSEUDOURIDINE MODIFICATIONS IN HUMAN tRNAs AND ARCHAEAL rRNAs MAJOR PROFESSOR: DR. RAMESH GUPTA RNAs undergo several post-transcriptional modifications inside the cell. The most abundant modification found in RNA is pseudouridine. Pseudouridine is present in all major classes of RNA. The classical TΨC sequence of tRNA reflects T (ribothymidine or 5-methyluridine) at position 54 in most Bacteria and Eukarya, and Ψ and C at positions 55 and 56, respectively, in nearly all tRNAs. TrmA and TruB homologs produce T54 and Ψ55, respectively, in Bacteria and Eukarya. However, archaeal tRNAs commonly have Ψ54 (or m1Ψ54) instead of T54, and Pus10 produces both Ψ54 and Ψ55 in these tRNAs. The pus10 gene is present in nearly all Archaea and most eukaryotes, but not in Bacteria and yeast. This coincides with the presence of Ψ54 in archaeal tRNAs and certain tRNAs (for Gln, Trp, Pro Thr, etc.) of animals, and its absence in the tRNAs of Bacteria and yeast. tRNAs for Trp and Pro that function as primers for replication of retroviruses also contain Ψ54. We found that Pus10 is the Ψ54 synthase in eukaryotes. The Ψ54 activity is specific for certain tRNAs, and it requires a conserved Am1AAU sequence at positions 57-60 of the tRNA for its maximum activity. Recombinant Pus10 can also form Ψ54 in select tRNAs and presence of m1A at position 58 is necessary for its maximum activity. Humans have two paralogs of TruB, TruB1, and TruB2 which are predicted to be the Ψ55 synthases for cytoplasmic and mitochondrial tRNAs, respectively. We found that recombinant human Pus10 can also modify Ψ55 of tRNAs in vitro. This Ψ55 activity of human Pus10 is not selective for specific tRNAs. Another pseudouridine synthase, Cbf5, which functions in guide dependent manner, is necessary for Ψ production in 23S rRNA of H. volcanii. Cbf5 is the catalytic component of the box H/ACA ribonucleoprotein complex that brings about these modifications. It consists of a guide RNA and three core proteins Nop10, Gar1, and L7Ae along with Cbf5. We found that Nop10 is necessary for Ψ production in 23S rRNA.

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CCA-adding enzymes synthesize and maintain the C-C-A sequence at the tRNA 3'-end, generating the attachment site for amino acids. While tRNAs are the most prominent substrates for this polymerase, CCA additions on non-tRNA transcripts are described as well. To identify general features for substrate requirement, a pool of randomized transcripts was incubated with the human CCA-adding enzyme. Most of the RNAs accepted for CCA addition carry an acceptor stem-like terminal structure, consistent with tRNA as the main substrate group for this enzyme. While these RNAs show no sequence conservation, the position upstream of the CCA end was in most cases represented by an adenosine residue. In tRNA, this position is described as discriminator base, an important identity element for correct aminoacylation. Mutational analysis of the impact of the discriminator identity on CCA addition revealed that purine bases (with a preference for adenosine) are strongly favoured over pyrimidines. Furthermore, depending on the tRNA context, a cytosine discriminator can cause a dramatic number of misincorporations during CCA addition. The data correlate with a high frequency of adenosine residues at the discriminator position observed in vivo. Originally identified as a prominent identity element for aminoacylation, this position represents a likewise important element for efficient and accurate CCA addition.

Η La πρωτεΐνη περιγράφηκε για πρώτη φορά στον άνθρωπο ως αυτοαντιγόνο το οποίο αναγνωρίζεται από αντισώματα που είναι παρόντα στον ορό ασθενών που πάσχουν από συστηματικό ερυθηματώδη λύκο (systemic lupus erythematosus) και σύνδρομο Sjögren. Είναι γνωστό σήμερα ότι συμμετέχει ενεργά στη βιογένεση μικρών μορίων RNA, συμπεριλαμβανομένου της ιδιότητάς της να συνδέεται και να προστατεύει την 3’ ακόλουθη αλληλουχία των πρόδρομων tRNA μεταγράφων. Επιπλέον, έχει προταθεί ότι in vivo διευκολύνει την απομάκρυνση της 5’ οδηγού αλληλουχίας από την RNase P. Στην παρούσα μελέτη, πραγματοποιήθηκε μοριακή κλωνοποίηση της La πρωτεΐνης του D. discoideum (354aa, 40,3-kDa) και υπερέκφρασή της. Η ανασυνδυασμένη La-His6 απομονώθηκε σε δύο στάδια καθαρισμού και εξετάστηκε ως προς την ικανότητα δέσμευσής της σε μόρια tRNA με ανάλυση EMSA (Electrophoresis Mobility Shift Assay). Τα αποτελέσματα με τη δοκιμή του ομόλογου pre-tRNASer που φέρει 3’ ακόλουθη αλληλουχία ως προσδέτη σε σχέση με ένα pre- tRNASer στο οποίο απουσιάζει η αντίστοιχη αλληλουχία, έδειξε ότι η La πρωτεΐνη έχει προτίμηση για αυτούσια πρόδρομα tRNA. In silico ανάλυση της πρωτεΐνης, έδειξε ότι περιλαμβάνει μοτίβα χαρακτηριστικά για RNA πρόσδεση με υψηλή συντήρηση στο Ν-τελικό της άκρο. Η προσθήκη αυξανόμενων συγκεντρώσεων ανασυνδυασμένης πρωτεΐνης La αναστέλλει σε ένα ποσοστό 10% την ωρίμανση του tRNA από την ομόλογη RNase P. Επιπρόσθετα, πραγματοποιήθηκαν in vitro δοκιμές για τη μελέτη της ικανότητας της La να βοηθά στη σωστή αναδίπλωση και άλλων πρόδρομων μεταγράφων της RNA πολυμεράσης ΙΙΙ. Για το λόγο αυτό ελέγχθηκε η ικανότητα ωρίμανσης παρουσία της La και της RNA υπομονάδας της RNase P. Κάτω από ποικίλες συνθήκες δοκιμής η παρουσία της La δεν ευνόησε την ενεργότητα του ριβοενζύμου. Τέλος, φωσφορυλίωση της La πρωτεΐνης σε συγκεκριμένα αμινοξέα δεν έδειξε ενίσχυση της συγγένειας για το υπόστρωμα. Η La του D. discoideum, μπορεί να συνδέεται με αρκετά μεγάλη συγγένεια με το ομόλογό της υπόστρωμα (Kd = 4±1nM, συγκρίσιμη με αντίστοιχες τιμές ομόλογων πρωτεϊνών) χωρίς την απαίτηση του μοτίβου 3’UUU-OH, το οποίο φαίνεται να είναι απαραίτητο για La πρωτεΐνες από άλλους οργανισμούς (H. sapiens, S. pombe, S. cerevisiae, T. brucei). Το γεγονός αυτό εγείρει πολλά ερωτήματα για την εξελικτική προέλευση αυτής της πρωτεΐνης και για την εξακρίβωση του ρόλου της ως αυτοαντιγόνου στα νοσήματα που αναφέρθηκαν. Βιοχημικές και δομικές μελέτες βρίσκονται σε εξέλιξη για να απαντήσουν σε αυτά τα ερωτήματα. / The La protein was first described in humans as an autoantigen recognized by antibodies present in serum of patients suffering from systemic lupus erythematosus and Sjögren syndrome. It is known that it participates actively in the biogenesis of small RNA molecules, including the binding and protection of the 3' trailer sequence of the precursor tRNA. Furthermore, it has been suggested that it facilitates the RNase P to remove the 5' leader sequence in vivo. In this study, molecular cloning and overexpression of the La protein D. discoideum (354aa, 40,3-kDa) were realized. Recombinant La-His6 was purified in two stages and was assayed regarding its capacity to bind tRNA using EMSA (Electrophoresis Mobility Shift Assay). The results testing the homologous pre- tRNASer bearing a 3' trailer sequence as a ligand compared to a pre- tRNASer which lacks the corresponding sequence, showed that the La protein has a preference for intact precursor tRNA. In silico analysis of the protein domain showed that it includes highly conserved motifs characteristic for RNA binding at the N-terminal site. The addition of increasing concentrations of recombinant La protein inhibits the maturation of tRNA by homologous RNase P at a 10% rate. Additionally, in vitro assays were carried out to study the ability of La to assist the proper folding of additional precursor transcripts of RNA polymerase III. Therefore the ability of maturation in presence of La and the RNA subunit of RNase P was assayed. Under various test conditions, the presence of La did not favor any riboenzyme activity. Finally, phosphorylation of La protein in specific amino acids showed no enhancing affinity to the substrate. The La protein from D. discoideum, can bind with quite a high affinity to its homologous substrate (Kd = 4 ± 1nM, comparable to corresponding values of homologous proteins) without the presence of the 3'UUU-OH motif, which appears to be necessary for La proteins from other organisms (H. sapiens, S. pombe, S. cerevisiae, T. brucei). This raises many questions about the evolutionary origin of this protein and the identification of its role as an autoantigen in many autoimmune diseases. Biochemical and structural studies are underway to answer these questions.

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