Plant luteoviral RNA viruses employ -1 frameshifting for the production of P1 and P1-P2 fusion proteins important for viral replication. Luteoviral pseudoknots are characterized by three adenosines in the 3' side of loop L2 known to be important for maintaining frameshifting efficiency and pseudoknot stability. A proposed P1-P2 mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) was of interest since it contained two adenosine to cytidine substitutions in L2. Functional analysis shows that the in vitro frameshifting efficiency is greater (~15%) than any other luteoviral pseudoknot. The NMR-derived solution structure of the ScYLV RNA pseudoknot shows that C25 is looped out of the triplex structure and the 3' most L2 cytidine (C27) and A24 form cis Watson-Crick/sugar-edge interactions with C14 and C15 in stem S1, respectively. Thus, the ScYLV pseudoknot maintains a similar triple helical architecture as other luteoviral pseudoknots. Surprisingly, the frameshifting efficiency of the C27A ScYLV pseudoknot is decreased by ~8 fold relative to wild-type ScYLV. The solution structure of the C27A ScYLV RNA exhibits a global fold similar to the wild-type RNA; however, distinct hydrogen bonding interactions at the helical junction are observed. Specifically, C8+ in the C8+ major groove base triple moves ~2.3 relative to the accepting (G12-C28) base pair relative to the WT RNA. New NMR experiments have been developed and/or applied to confirm Watson-Crick base pairs and tertiary structural interactions in the PEMV-1 and ScYLV pseudoknots by direct observation of trans hydrogen bond scalar couplings. In addition, intrabase couplings in cytidine and adenosine have been measured, providing a valuable tool for the assignment of amino and N3/N1 resonances in RNA. Finally, thermodynamic analysis of the pairwise coupling between the major groove and minor groove tertiary structural hydrogen bonds at the helical junction have been investigated by monitoring the thermal unfolding of WT, dC14, C27A, and dC14/C27A RNAs as a function of pH. Favorable pairwise coupling characterized the WT ScYLV and BWYV RNAs, while unfavorable coupling characterized the poorly functional C27A ScYLV RNA. The implications of these structural, functional, and thermodynamic findings on the mechanism of frameshift stimulation is discussed.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18334号 / 農博第2059号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4841(農学部図書室) / 31192 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 奥野 哲郎, 教授 佐久間 正幸, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity. First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys. Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact. Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described. A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C. cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent. During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

We developed a new program called GeneTack for ab initio frameshift detection in intronless protein-coding nucleotide sequences. The GeneTack program uses a hidden Markov model (HMM) of a genomic sequence with possibly frameshifted protein-coding regions. The Viterbi algorithm nds the maximum likelihood path that discriminates between true adjacent genes and a single gene with a frameshift. We tested GeneTack as well as two other earlier developed programs FrameD and FSFind on 17 prokaryotic genomes with frameshifts introduced randomly into known genes. We observed that the average frameshift prediction accuracy of GeneTack, in terms of (Sn+Sp)/2 values, was higher by a signicant margin than the accuracy of the other two programs. GeneTack was used to screen 1,106 complete prokaryotic genomes and 206,991 genes with frameshifts (fs-genes) were identifed. Our goal was to determine if a frameshift transition was due to (i) a sequencing error, (ii) an indel mutation or (iii) a recoding event. We grouped 102,731 genes with frameshifts (fs-genes) into 19,430 clusters based on sequence similarity between their protein products (fs-proteins), conservation of predicted frameshift position, and its direction. While fs-genes in 2,810 clusters were classied as conserved pseudogenes and fs-genes in 1,200 clusters were classied as hypothetical pseudogenes, 5,632 fs-genes from 239 clusters pos- sessing conserved motifs near frameshifts were predicted to be recoding candidates. Experiments were performed for sequences derived from 20 out of the 239 clusters; programmed ribosomal frameshifting with eciency higher than 10% was observed for four clusters. GeneTack was also applied to 1,165,799 mRNAs from 100 eukaryotic species and 45,295 frameshifts were identied. A clustering approach similar to the one used for prokaryotic fs-genes allowed us to group 12,103 fs-genes into 4,087 clusters. Known programmed frameshift genes were among the obtained clusters. Several clusters may correspond to new examples of dual coding genes. We developed a web interface to browse a database containing all the fs-genes predicted by GeneTack in prokaryotic genomes and eukaryotic mRNA sequences. The fs-genes can be retrieved by similarity search to a given query sequence, by fs- gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, programmed frameshifts etc. All the tools and the database of fs-genes are available at the GeneTack web site http://topaz.gatech.edu/GeneTack/

The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.