Two plaque-pure phage lambda clones designated as λhtX-l and λhtX-2 that hybridized to unfractionated bovine liver tRNA were isolated from a human X chromosome-specific library. The λDNAs were characterized by restriction mapping and Southern blot hybridization techniques. The human DNA segment in λhtX-l contains five or more presumptive tRNA genes and at least one Alu family member. The 19-kilobase human DNA insert in λhtX-2 contains two or more presumptive tRNA genes and at least three Alu family members. Another human genomic clone designated λhVKV7 hybridized to mammalian valine tRNA IAC. The clone was characterized by physical mapping and Southern blot hybridization techniques. The 18.5-kilobase human DNA fragment in λhVKV7 contains a cluster of three tRNA genes and at least nine Alu family members.

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37°C for 20 min, with protein at 180 μg ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.

An 18.5-kb human DNA segment was selected from a human XCharon-4A library by hybridization to mammalian valine tRNAiAc and found to encompass a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNAcuu gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region of the human DNA insert. At least nine Alu family members were found interspersed throughout the human DNA fragment. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase Ti fingerprints of the mature-sized tRNA transcription products are consistent with the DNA sequences of the structural genes. Three members of the chimpanzee triosephosphate isomerase (TPI) gene family, the functional transcription unit and two processed pseudogenes, were characterized by genomic blotting and DNA sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first complete hominoid TPI gene sequenced. The gene exhibits a very high identity with the human and rhesus TPI genes. In particular, the polypeptides of 248 amino acids encoded by the chimpanzee and human TPI genes are identical, although the two coding regions differ in the third codon wobble positions for five amino acids. An Alu member occurs upstream from one of the processed pseudogenes, whereas an isolated endogenous retroviral long terminal repeat (HERV-K) occurs within the structural region of the other processed pseudogene. The ages of the processed pseudogenes were estimated to be 2.6 and 10.4 million years, implying that one was inserted into the genome before the divergence of the chimpanzee and human lineages, and the other inserted into the chimpanzee genome after the divergence.

Ribosomes are universally conserved macromolecular machines found within all living cells that catalyze protein synthesis, one of nature’s most fundamental processes. Ribosomes synthesize proteins, which are polymeric chains of amino acids, by incorporating the amino acids one at a time via aminoacylated-transfer RNAs (aa-tRNAs), based on translation of the sequence of triplet- nucleotide codons presented by the messenger RNA (mRNA) template that is a direct readout of genomic DNA. Recent biochemical, structural, dynamic, and computational studies have uncovered large-scale conformational changes of the ribosome, its tRNA substrates, and the additional protein translation factors that play important roles in regulating protein synthesis, especially during the elongation phase of translation when the bulk of each protein is synthesized. How the ribosome, its translation elongation factors, tRNAs, and mRNA physically coordinate and regulate the movements of the tRNAs carrying amino acids into, through, and out of the ribosome remains one of the more fundamental questions in the mechanistic studies of protein synthesis. A complete understanding of the conformational dynamics of ribosomal complexes will improve our knowledge of how translation is regulated, including how ribosome-targeting antibiotics regulate translation elongation, and will provide crucial information for designing next-generation antibiotics. In this thesis I have investigated the conformational dynamics of the ribosome during the elongation phase of protein synthesis at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET) microscopy experiments. Specifically, I have studied ribosomal dynamics during the elongation phase of translation in the presence of a tRNAPro in the context of an mRNA that has the propensity to shift out of the reading frame. My studies have revealed information about the mechanistic and regulatory functions of the posttranscriptional modifications of tRNAPro in a context in which the ribosomal complex has the propensity to undergo non-programmed +1-frameshifting, in which the tRNA-mRNA base pairing shifts one base toward the 3’ end of the mRNA, and if unchecked, leads to the synthesis of a polypeptide with a completely different sequence of amino acids. My data suggests that in this context, the mechanism underlying non-programmed +1-frameshifting involves the tRNA shifting out of frame prior to the tRNA being accommodated in the P site, i.e. either while the tRNA is in the A site, or more likely, during translocation of the tRNA from the A site to the P site, and not while the tRNA is already occupying the P site, as previously proposed.

Protein synthesis in archaebacteria and the cytoplasm of eukaryotes is initiated using the initiator methionyl-tRNA (Met-tRNA[subscript i][superscript Met]). In contrast, formylated methionyltRNA (fMet-tRNA[subscript i][superscript Met][subscript f]) is found in eubacteria, and in chloroplasts and mitochondria of eukaryotes, and this formylated initiator tRNA was widely believed to be required for initiation of protein synthesis in those systems. However, the fact that initiation of protein synthesis in yeast mitochondria can occur with unformylated initiator tRNA has changed our perspective about the initiation of mitochondrial protein synthesis. This dissertation is composed of two parts. Part I describes an investigation of the yeast AEP3 gene which was isolated by a genetic screening system in Saccharomyces cerevisiae. The main goal of this part was to discover new accessory factor(s) that might be involved in initiation of protein syntheis of yeast mitochondria when there is no formylation of initiator tRNA and determine how they support the initiation process in Saccharomyces cerevisiae. The synthetic petite genetic screen identified the AEP3 gene. Protein-protein binding assays as well as protein-initiator tRNA binding assays indicate that Aep3p is associated with the initiation process in yeast mitochondrial protein synthesis. This discovery is important because it suggests the possible mechanism by which initiation of protein synthesis in yeast mitochondria occur under conditions where there is no formylation of initiator tRNA. Part II describes a study of the TRM5 gene encoding a tRNA methyltransferase in S. cerevisiae. The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) previously known to methylate guanosine at position 37 (m¹G37) in certain cytoplasmic tRNAs in S. cerevisiae. The main goal of this part was to investigate whether Trm5p is also responsible for m¹G37 modification of mitochondrial tRNAs. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a [Delta]trm5 mutant strain, as well as with a synthetic mitochondrial tRNA[superscript Met][subscript f] and tRNA[superscript Phe]. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Analysis of subcellular fractionation and immunoblotting revealed that the enzyme was localized to both cytoplasm and mitochondria. Our data including the analysis of N-terminal truncation mutants suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis. / text

Over the last twenty years, the expansion of the genetic code has been made possible by the encoding of unnatural amino acids into proteins. Unnatural amino acids could be used to expand the chemical functionalities available to biology allowing for the production of ‘allo-proteins’ with potentially novel structures and functions. One method to engineer the genetic code is to engineer the translational components responsible for its maintenance. This methodology relies primarily on the evolution of the aminoacyl tRNA synthetases and their cognate tRNAs to produce an orthogonal enzyme and tRNA pair that allows for the insertion of unnatural amino acids into proteins. To date only a handful of these orthogonal pairs are available for use in genetic code expansion. As in vitro and in vivo techniques to re-code the genetic code have expanded, the utility of having multiple orthogonal pairs to site-specifically insert multiple unnatural amino acids into proteins has increased. In addition, the development of a variety of orthogonal pairs based on the twenty canonical aminoacyl tRNA synthetase-tRNA pairs will expand the types of unnatural amino acid sidechains available for protein engineering efforts. Herein we describe the engineering of the tryptophanyl tRNA synthetase and tRNA superscript Trp], pair from yeast for use as an orthogonal pair in E. coli. We have successfully built and tested synthetic expression constructs for the expression of this orthogonal pair in vivo. In addition, we have rationally engineered an orthogonal amber nonsense suppressor tRNA based on the yeast tRNA[superscript Trp], dubbed AS3.4. This suppressor has been shown to be an efficient orthogonal suppressor tRNA in vivo, and will aid in our efforts to expand the genetic code with heterocyclic unnatural amino acids. We also have developed a potentially tunable two part selection scheme, for use in the directed evolution of mutant tRNA synthetases that are specific to unnatural amino acid substrates. / text

Thesis (Ph.D.)--Ohio University, March, 2007. / Includes bibliographical references (leaves 190-200)

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008. / Committee Chair: Harvey, Stephen C; Committee Member: Hud, Nicholas V; Committee Member: Oyelere, Adegboyega; Committee Member: Wartell, Roger.