The 7-deazaguanosine nucleosides queuosine (Q) and archaeosine (G⁺) are two of the most structurally complex modified nucleosides found in tRNA. Q is found exclusively in the wobble position of tRNAGUN coding for the amino acids asparagine, aspartate, histidine and tyrosine in eukarya and bacteria, while (G⁺) occurs in nearly all archaeal tRNA at position 15. In archaea preQ₀ is inserted into tRNA by the enzyme tRNA-guanine transglycosylase (TGT), which catalyzes the exchange of guanine with preQ₀ to produce preQ₀-tRNA. The first objective of this study was to identify and characterize the enzyme(s) catalyzing the conversion of preQ₀-tRNA to G+-tRNA. Comparative genomics identified a protein family possibly involved in the final steps of archaeosine biosynthesis, which was annotated as TgtA2. Structure based alignments comparing TGT and TgtA2 revealed that TgtA2 lacked key TGT catalytic residues and contained an additional module. The gene corresponding to "tgtA2" from "Methanocaldococcus jannaschii (mj1022)" was cloned, expressed and the purified recombinant enzyme characterized. Recombinant MjTgtA2 was shown to convert preQ₀-tRNA to G⁺-tRNA using glutamine, asparagine or NH₄⁺ as nitrogen donors in an ATP-independent reaction. This is the only example of the conversion of a nitrile to a formamidine known in biology. QueF catalyzes the reduction of preQ₀ to 7-aminomethyl-7-deazaguanine preQ₀ in the queuosine biosynthetic pathway. The second aim of this study was the transient state kinetic analysis of substrate binding and catalysis by the enzyme QueF, as well as investigation of the effects of ligands on its quaternary structure. Gel filtration and sedimentation equilibrium analyses indicated that QueF exists as a hybrid population in a rapid equilibrium between decamer and pentamer states. Addition of preQ₀ to QueF resulted in shifting the equilibrium towards the decamer state, as did the addition of divalent metals. Potassium chloride at high concentrations was found to disrupt the quaternary structure of QueF. Intrinsic tryptophan and NADPH fluorescence was used to determine the substrate binding to QueF by stopped-flow kinetic studies. Studies on the binding of preQ₀ to QueF in conjuction with binding NADPH to the QueF mutant E78A-thioimide intermediate suggested a two-step mechanism consisting of a fast bimolecular process and a subsequent slower unimolecular process, while the binding of preQ₀ to the C55A mutant was monophasic, consisting of only the fast bimolecular process. Thioimide formation was monitored by UV-Vis; under single turnover conditions the data fit well to single exponential rise. However, at high preQ₀ concentrations two phases could be observed. The reduction of the thioimide was determined under single turnover conditions by both UV-Vis and fluorescence, and comparable rates were obtained from both techniques. These results indicate that the binding of preQ₀ and NADPH to QueF, as well as thioimide formation, are very rapid; and that reduction of the thioimide is most likely the rate limiting step. Analysis of component rates suggests structural changes occur between these steps, further limiting the overall rate.

No description available.

In order to expand our understanding of the expression of chloroplast genes, the structure and expression of the Euglena gracilis rpoC1 and rpoC2 loci were studied. The rpoC1 and rpoC2 gene products are similar to the amino- and carboxyl-terminal regions of the $\beta\sp\prime$ subunit of E. coli RNA polymerase. The nucleotide sequence (7,270 bp) was determined for 100% of both strands encoding these two genes. The rpoC1 and rpoC2 genes are located downstream and in the same polarity as the rpoB gene. The organization of the Euglena rpoB-rpoC1-rpoC2 genes is conserved in plant chloroplasts and is similar to the E. coli rpoB-rpoC operon. The Euglena rpoC1 gene (586 codons) encodes a polypeptide with a predicted molecular weight of 68,043. The rpoC1 gene is interrupted by one group II intron of 349 bp, seven group III introns of 107, 100, 119, 97, 110, 102 and 103 bp, and three atypical introns of 210, 213 and 198 bp. The Euglena rpoC2 gene (830 codons) encodes a polypeptide with a predicted molecular weight of 94,628. The rpoC2 gene is interrupted by two group II introns of 580 and 514 bp, respectively. All of the exon-exon junctions were experimentally determined via cDNA cloning and sequencing analysis. Multiple protein alignments of the rpoC1 and rpoC2 gene products with related proteins from bacteria and chloroplasts were used to identify conserved regions. Transcripts from the rpoC1 and rpoC2 loci were characterized via Northern analysis. The rpoB, rpoC1 and rpoC2 genes are cotranscribed. Fully spliced tri-, di- and monocistronic transcripts were detected with hybridization probes specific for each gene. The relative abundance of the rpoC1 and rpoC2 transcripts is similar in RNA from dark- and light-grown Euglena. The mature 5'-ends of the rpoC1 and rpoC2 genes were mapped by primer extension. The 3'-end of the mature rpoC2 transcript was localized via an S1 nuclease protection assay. The rpoC1 and rpoC2 gene products were also compared to the largest subunits of RNA polymerases from archaebacteria and eukaryotes. The evolution of the Euglena genes is discussed.

Protein synthesis, one of nature's most fundamental processes within all living cells, is catalyzed by the ribosome, a highly conserved, massive, two-subunit ribonucleoprotein complex. Ribosomes synthesize proteins based on the sequence of triplet-nucleotide codons presented by the messenger RNA (mRNA) template, using aminoacyl-transfer RNAs (aa-tRNAs) substrates, which deliver individual amino acids to the ribosome. Recent biochemical, structural, dynamic and computational studies have uncovered large-scale conformational changes of the ribosome, its tRNA substrates, and translation factors that play important roles in regulating protein synthesis, especially during the elongation phase of translation. For example, translocation of the ribosome along its mRNA template involves several conformational rearrangements of the ribosomal pre-translocation (PRE) complex, including the rotation of two ribosomal subunits, closure of the L1 stalk element, and reconfigurations of the ribosome-bound tRNAs. Importantly, modulation of these conformational changes of PRE complexes is used as a strategy by the cell and ribosome-targeting antibiotics to regulate translation elongation. Therefore, a complete understanding of the conformational dynamics of ribosomal complexes will not only improve our knowledge on how translation is regulated, but also provide crucial information for designing next-generation antibiotics. This thesis presents efforts demonstrating several strategies the cell develops in order to regulate translation by modulating the conformational dynamics of ribosomal complexes. In Chapter 2, I investigate if and how the individual dynamics of intersubunit motion, tRNA and L1 stalk are coordinated within PRE complexes, so that the translocation reaction is facilitated. To address this question, the dynamics of ribosomal intersubunit rotation were predictably perturbed using either structurally guided ribosome mutagenesis as well as an ribosome-targeting antibiotic translation inhibitor. Correspondingly, I used two single-molecule fluorescence resonance energy transfer (smFRET) signals to directly monitor how perturbation of the dynamics of intersubunit rotation alter the dynamics of P-site tRNA and the L1 stalk in PRE complexes. Taken together with the results of my complementary in vitro biochemical assays, my smFRET work clearly demonstrates that the ribosome coordinates individual conformational changes to maximize and regulate the efficiency of the translocation reaction. It is very likely that this strategy is used by the ribosome in other steps during translation for efficient chemical or mechanical reactions, and is taken advantage of by translation factors and antibiotics as part of the mechanisms through which they regulate and inhibit translation, respectively. Energy-dependent translational throttle A (EttA) is one regulatory translation factor that has been recently discovered and characterized through a collaboration between the Hunt, Gonzalez, and Frank laboratories (Chapter 3). Biochemical experiments have shown that in the presence of a high ADP/ATP ratio, EttA inhibits formation of the first peptide bond, and such inhibition is relieved upon addition of ATP, indicating that EttA may regulate the synthesis of proteins in response to the energetic status of the cell, as reflected by the cellular ADP/ATP ratio. Complementary cryo-EM studies have shown that the ATP-bound form of EttA binds to the ribosome at the E-site from where it directly contacts and forms bridging interaction between the L1 stalk and P-site tRNA. The results of my smFRET experiments demonstrate that EttA differentially modulates the conformation and/or dynamics the L1 stalk, depending on whether EttA is bound to ADP or ATP, thereby providing a possible rationale for the distinct effects of EttA on dipeptide synthesis in the presence of ADP vs. ATP. My smFRET data, together with the biochemical and structural efforts, demonstrate that EttA functions, at least in part, by restricting ribosome and tRNA dynamics that are crucial for translation. More importantly, our data support a model for the interaction of EttA with the ribosomal complex and its regulation of translation at the start of the elongation cycle, the molecular mechanism of which EttA uses has never been found among all other known translational regulatory factors. +1 non-programmed ribosomal frameshifting (+1 FS), in which the elongating ribosome slips by one nucleotide towards the 3' end of the mRNA during translation, occurs at a low frequency as a translational error. Proline-tRNA with an anticodon GGG (tRNAProGGG) is prone to induce +1 FS, and the evolution of post-transcriptional modifications of tRNAProGGG is used as a strategy by nature to suppress this error. However, the mechanism underlying this suppression has not been previously characterized. tRNAProGGG modifications have been shown to play important roles in regulating the conformational stability and flexibility of the secondary and tertiary structures of tRNAs and therefore have the potential to regulate the conformational dynamics of ribosomal complexes during translation elongation. However, the effect of these modifications on elongating ribosomal complex dynamics is completely unknown, thus greatly impeding our understanding of the role that they play in maintaining the translational reading frame. Chapter 4 presents the efforts to elucidate the mechanism by which tRNA modifications suppress +1 FS by investigating the effects that modifications of tRNAProGGG have on regulating the conformational dynamics of ribosomal complexes in a collaboration with the Hou group at Thomas Jefferson University. The preliminary results from my smFRET experiments suggest that tRNAProGGG modifications do indeed play a role in modulating the dynamics of ribosomal complexes. More importantly, the combination of tRNA without modifications and mRNA carrying a sequence that is prone to induce +1 FS dramatically alters ribosome dynamics, probably by affecting tRNA flexibility, tRNA-ribosome, tRNA-mRNA, and mRNA-ribosome interactions, all of which could have important implications for how +1 FS occurs.

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 105-123). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 119-129). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 119-129). Also available on the Internet.

Not available / text

The rpoB gene coding for a β-like subunit (homologous to the E. coli DNA-dependent RNA polymerase β subunit) of the chloroplast DNA-dependent RNA polymerase was located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. The complete nucleotide sequence of the gene was determined. The sequence includes 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resemble the E. coli rpoB-rpoC and higher plants chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp, respectively, and a Group II intron of 309 bp. All other known chloroplast rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNAs, as well as rpoB-rpoC1 and rpoB-rpoC1-rpoC2 mRNAs were identified. Unspliced intron-containing transcripts could not be detected in these experiments. The rpoB gene is the first gene in the RNA polymerase rpoB-rpoC1-rpoC2 transcription unit. The three genes are transcribed from a promoter located upstream the rpoB gene. The transcript is processed to mature monocistronic mRNAs. The relative abundance of the mono-, di- and tricistronic mRNAs appear to be similar in RNAs isolated from photoautotrophic, heterotrophic and dark grown cells. The mature 5'- and 3'-ends of the mature rpoB monocistronic transcripts were determined via S1 nuclease mapping and primer extension RNA sequencing. In addition, the sequence of the 23S rRNA from the rrnC operon and the intergenic spacer between the rrnA and rrnB operon were determined. Transcription initiation for the ribosomal RNA transcription unit was determined via Northern analysis and S1 nuclease mapping of chloroplast RNA that was in vitro 5'-end labeled. Two transcription initiation sites were mapped at positions +1 and -50 upstream the 16S rRNA gene. The 3'-ends of the rrnA/rrnB and rrnC 5S rRNA were determined using S1 nuclease protection experiments. The protected fragments were of identical size. The rpoB-C1-C2 DNA sequence has been submitted to EMBL, accession number X17171, and the 23S rRNA DNA sequence was given the number X13310.