tRNA genes have the fundamental role of translating the genetic code during protein synthesis. Beyond solely a passive decoding role, the tRNA pool exerts selection pressures on the codon usage of organisms and the viruses that infect them because processing codons read by rare tRNAs can be slow or even erroneous. To better understand the interactions of codons and anticodons in eukaryotic species, we first investigated whether tRNAs packaged into HIV-1 particles may relate to the poor codon usage of HIV-1 genes. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding poorly adapted codons are overrepresented in HIV-1 virions. Because the affinity of Gag-Pol for all tRNAs is non-specific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Moreover, differences that we found in the codon usage between early and late genes suggest alterations in the tRNA pool are induced late in viral infection. Next, we tested whether a reduced tRNA anticodon pattern, which was called into question by predicted tRNA datasets, is maintained across eukaryotes. tRNA prediction methods are prone to falsely identifying tRNA-derived repetitive sequences as functional tRNA genes. Thus, we proposed and tested a novel approach to identify falsely predicted tRNA genes using phylogenetics. Phylogenetic analysis removed nearly all the genes deviating from the anticodon pattern, therefore the anticodon pattern is reaffirmed across eukaryotes.

tRNA genes have the fundamental role of translating the genetic code during protein synthesis. Beyond solely a passive decoding role, the tRNA pool exerts selection pressures on the codon usage of organisms and the viruses that infect them because processing codons read by rare tRNAs can be slow or even erroneous. To better understand the interactions of codons and anticodons in eukaryotic species, we first investigated whether tRNAs packaged into HIV-1 particles may relate to the poor codon usage of HIV-1 genes. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding poorly adapted codons are overrepresented in HIV-1 virions. Because the affinity of Gag-Pol for all tRNAs is non-specific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Moreover, differences that we found in the codon usage between early and late genes suggest alterations in the tRNA pool are induced late in viral infection. Next, we tested whether a reduced tRNA anticodon pattern, which was called into question by predicted tRNA datasets, is maintained across eukaryotes. tRNA prediction methods are prone to falsely identifying tRNA-derived repetitive sequences as functional tRNA genes. Thus, we proposed and tested a novel approach to identify falsely predicted tRNA genes using phylogenetics. Phylogenetic analysis removed nearly all the genes deviating from the anticodon pattern, therefore the anticodon pattern is reaffirmed across eukaryotes.

tRNA genes have the fundamental role of translating the genetic code during protein synthesis. Beyond solely a passive decoding role, the tRNA pool exerts selection pressures on the codon usage of organisms and the viruses that infect them because processing codons read by rare tRNAs can be slow or even erroneous. To better understand the interactions of codons and anticodons in eukaryotic species, we first investigated whether tRNAs packaged into HIV-1 particles may relate to the poor codon usage of HIV-1 genes. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding poorly adapted codons are overrepresented in HIV-1 virions. Because the affinity of Gag-Pol for all tRNAs is non-specific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Moreover, differences that we found in the codon usage between early and late genes suggest alterations in the tRNA pool are induced late in viral infection. Next, we tested whether a reduced tRNA anticodon pattern, which was called into question by predicted tRNA datasets, is maintained across eukaryotes. tRNA prediction methods are prone to falsely identifying tRNA-derived repetitive sequences as functional tRNA genes. Thus, we proposed and tested a novel approach to identify falsely predicted tRNA genes using phylogenetics. Phylogenetic analysis removed nearly all the genes deviating from the anticodon pattern, therefore the anticodon pattern is reaffirmed across eukaryotes.

tRNA genes have the fundamental role of translating the genetic code during protein synthesis. Beyond solely a passive decoding role, the tRNA pool exerts selection pressures on the codon usage of organisms and the viruses that infect them because processing codons read by rare tRNAs can be slow or even erroneous. To better understand the interactions of codons and anticodons in eukaryotic species, we first investigated whether tRNAs packaged into HIV-1 particles may relate to the poor codon usage of HIV-1 genes. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding poorly adapted codons are overrepresented in HIV-1 virions. Because the affinity of Gag-Pol for all tRNAs is non-specific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Moreover, differences that we found in the codon usage between early and late genes suggest alterations in the tRNA pool are induced late in viral infection. Next, we tested whether a reduced tRNA anticodon pattern, which was called into question by predicted tRNA datasets, is maintained across eukaryotes. tRNA prediction methods are prone to falsely identifying tRNA-derived repetitive sequences as functional tRNA genes. Thus, we proposed and tested a novel approach to identify falsely predicted tRNA genes using phylogenetics. Phylogenetic analysis removed nearly all the genes deviating from the anticodon pattern, therefore the anticodon pattern is reaffirmed across eukaryotes.

No description available.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Le travail retranscrit dans cette thèse regroupe l'étude de différents processus biologiques impliquant les ARN de transfert. Premièrement, dans le cadre de l'étude du rôle de la protéine de nucléocapside (NC) dans la formation du complexe d'initiation de la transcription inverse du VIH-1, un site de fixation fort et spécifique de la NC a été identifié sur le bras D de l'ARNtLys3 — l'ARNt servant d'amorce à la transcriptase inverse lors de la synthèse du brin d'ADNc(-). Cette fixation permet d'une part la fusion des interactions tertiaires entre les bras T et D de l'ARNtLys3 et pourrait d'autre part être l'un des facteurs déplaçant l'équilibre vers la formation de l'hybride ARNtLys3/ARN viral. L'étude structurale par RMN du complexe NC/bras D s'est heurtée à des problèmes d'échange chimique dans la gamme intermédiaire–rapide de l'échelle de temps spectrale. L'utilisation de séquences de type CPMG–HSQC a permis d'améliorer le rapport signal/bruit des expériences RMN, et particulièrement celui des signaux des résidus de l'interface. Ceci nous a permis d'identifier les résidus de la NC impliqués dans la reconnaissance du bras D. Deuxièmement, la structure de la m1A58 méthyltransférase de T. thermophilus (TrmI) — une enzyme de modification des ARNt — a été résolue par cristallographie et affinée à 1.7 Å. Ceci a permis d'identifier trois résidus potentiellement impliqués dans la catalyse et/ou la reconnaissance du substrat ARNt (D170, Y78 et Y194). La production de variants de TrmI au niveau de ces résidus ainsi que des études d'amarrage moléculaire d'une adénine au site actif ont permis de confirmer cette hypothèse et de proposer un rôle catalytique pour chacun d'eux. Parallèlement, des études par gel natif et par spectrométrie de masse en conditions non dénaturantes ont montré une stoechiométrie 1/2 pour le complexe entre l'enzyme TrmI tétramérique et le substrat ARNt. Troisièmement, la résolution de la structure cristallographique de l'ARNtfMet initiateur d'E. coli a révélé une conformation unique de la région de l'anticodon. Cette conformation unique est associée au paires GC de la tige anticodon, caractéristiques des ARNt initiateurs. Cette conformation particulière — dans laquelle la base A37 ne vient pas s'empiler entre les bases 36 et 38, comme dans tous les ARNt élongateurs — permet de mettre en lumière de nombreux résultats biochimiques de la littérature et suggère un mécanisme par lequel la machinerie de l'initiation de la traduction pourrait discriminer l'ARNt initiateur de tous les ARNt cellulaires.

Killer strains of the yeast Kluyveromyces lactis secrete a heterotrimeric protein toxin (zymocin) to inhibit the growth of sensitive yeasts. The cytotoxicity of zymocin resides in the γ subunit (γ-toxin), however the mechanism of cytotoxicity caused by γ-toxin was previously unknown. This thesis aimed to unravel the mode of γ-toxin action and characterize the interaction between γ-toxin and its substrates. Previous studies suggested a link between the action of γ-toxin and a distinct set of transfer RNAs. In paper I, we show that γ-toxin is a tRNA anticodon endonuclease which cleaves tRNA carrying modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at position 34 (wobble position). The cleavage occurs 3’ to the wobble uridine and yields 2’, 3’-cyclic phosphate and 5´-hydroxyl termini. In paper II, we identified the determinants in tRNA important for efficient γ-toxin cleavage. The modifications present on the wobble uridines have different effects on tRNA cleavage by γ-toxin. The Saccharomyces cerevisiae wobble modification mcm5 group has a strong positive effect, whereas the Escherichia coli wobble modification 5-methylaminomethyl group has a strong inhibitory effect on tRNA cleavage. The s2 group present in both S. cerevisiae and E. coli tRNAs has a weaker positive effect on the cleavage. The anticodon stem loop (ASL) of tRNA represents the minimal structural requirement for γ-toxin action. Nucleotides U34U35C36A37C38 in the ASL are required for optimal cleavage by γ-toxin, whereas a purine at position 32 or a G at position 33 dramatically reduces the reactivity of ASL. Screening for S. cerevisiae mutants resistant to zymocin led to the identification of novel proteins important for mcm5s2U formation (paper III). Sit4p (a protein phosphatase), Sap185p and Sap190p (two of the Sit4 associated proteins), and Kti14p (a protein kinase) are required for the formation of mcm5 side chain. Ncs2p, Ncs6p, Urm1p, and Uba4p, the latter two function in a protein modification (urmylation) pathway, are required for the formation of s2 group. The gene product of YOR251C is also involved in the formation of s2 group. The involvement of multiple proteins suggests that the biogenesis of mcm5s2U is very complex.

No description available.