Protein synthesis is an essential process for all living organisms and is an effective major target for current antibiotics. Elongation factor Tu (EF-Tu) is a highly conserved and essential protein that functions during protein synthesis. EF-Ts interacts with EF-Tu to help maintain a functionally active state of EF-Tu required for cell growth. Although EF-Ts is essential for Escherichia coli, its sequence is poorly conserved. LepA is a highly conserved protein within bacteria and has a similar structure to EF-Tu. In spite of this, LepA has been shown to be non-essential under ideal conditions and the function of LepA still remains elusive. An analysis on the structurally unique aspects of LepA, EF-Tu and EF-Ts was performed here in an effort to gain an understanding on the functions of these proteins. This knowledge, in combination with their unique structural components will provide important tools in developing new and effective antibiotics. / xiii, 177 leaves : col. ill. ; 29 cm

A thesis submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, South Africa, in fulfilment of the requirements for the Degree of Doctor of Philosophy, This thesis presented as a series of manuscripts 1993 / In this study the morphologic, cytokinetic, biochemical, and molecu1w: consequences of low-dose continuous Puromycin-exposure were examined in HL-60 cells, and in a variety of malignant and non-malignant human and murine cell types. Puromycin (PM) is a composite of the amino nucleoside dimethyladenosine and tyrosine-o-methylether. Functionally it is an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, more specifically of tyrosyl..tRNA. At high concentrations 5-S0#tg/ml (10-100#tM) PM has been found to block protein synthesis completely by causing the premature release from the ribosomes of truncated peptide chains which are bound to PM through their carboxyl termini. The nascent PMGpeptide complexes (PMPs) are rapidly degraded through a ubiquitin-dependent pathway and are of interest because of (i) their potential to compete for degradation with the natural substrates of the ubiquitin-dependent pathway, including cyclin B, and (ii) because their structure predicts an inhibitory effect on tyrosine kinase activity. In the current' study then, special consideration was given to the effect of PM on the cell cycle, on apoptosis (programmed. cell death), and on tyrosine kinase activity, As a means of comparison, certain of these effects were also examined with respect to another translation inhibitor Cycloheximide (CHX), to two other substituted purines Puromycin Amino nucleoside (PAN) and 6..Dimethylaminopurine (6-DMAP), as well as to the cyclophospbamide derivative Mafosfamide (ASTA Z 7557). / GR2017

Translation is the process by which the cell produces new proteins on the ribosome, as directed by genetic instructions, in all living organisms. Structural studies of the ribosome have shed considerable lights on its mechanism and regulation. Cryogenic electron microscopy (cryo-EM) and single-particle reconstruction technique is one of the major approaches to studying ribosome structure. In this thesis, I report the use of cryo-EM and related new techniques to study the structure of ribosome complexes. This work is divided into three parts. First, in Chapter 3, I describe the development of a computational method in the classification of cryo-EM data. Recently developed classification methods have enabled resolving multiple structures/conformations of the molecules from cryo-EM data obtained on a heterogeneous biological sample. However, the classification methods all involve various amounts of arbitrary decisions made by researchers, which can limit the use of these methods by inexperienced users. As a step toward fully automated classification, I worked with colleagues to develop a "jumper analysis" to determine the number of distinguishable classes of 3D reconstruction, based on the statistics of cryo-EM particles. Second, in Chapter 4, I document the cryo-EM study of EttA-70S ribosome complex, which provided structural insights into the mechanism of EttA in translation regulation. Energy-dependent translation throttle A (EttA, previously named YjjK in Escherichia coli) is the most prevalent member of ATP-binding cassette F family proteins in eubacteria. Through a collaboration among the Hunt, Frank, and Gonzalez labs, we combined crystallography, biochemical, cryo-EM and single-molecule fluorescence energy transfer techniques to elucidate the function and mechanism of EttA. We demonstrated that EttA gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. We also showed that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, and restricts the ribosome and tRNA dynamics required for translation. Thirdly, in Chapter 5, I discuss the improvements to a new technique, time-resolved cryo-EM by mixing-spraying, and its application to ribosome studies. The mixing-spraying method can study processes involving two big biological molecules that are in the sub-second time scale. I worked with colleagues to apply this method to studying ribosome subunit association. By mixing the subunits and reacting for 60 ms and 140 ms, we were able to capture the association reaction in a pre-equilibrium state. We detected three 70S ribosome conformations in the system. Quantification of the proportions of particles assuming these conformations suggested that the 70S ribosome can undergo fast conformational changes upon formation, and reaches equilibrium among these conformations earlier than 60 ms. In addition, I present preliminary results of studying translation decoding using the mixing-spraying method. This study, performed before improving the mixing-spraying method, was inconclusive mainly due to the limited size of cryo-EM data. Now that we have demonstrated the capability of the mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, the translation decoding process can be revisited and many other processes, such as translation initiation, can be studied.

Dendritic cells are a key population in the immune system. There are two main subsets: conventional DC (sometimes called classical DC, cDCs), which survey tissues for pathogens and activate naive T cells, and plasmacytoid dendritic cells (pDCs), which produce high amounts of IFNα in response to viral products. Both subsets begin development in the bone marrow from common dendritic progenitor (CDP), and driven by Flt3 signaling induced by the growth factor Flt3L. The CDP gives rise to a cDC committed preDC which exits the bone marrow and seeds peripheral organs to give rise to cDCs, while pDCs finish development in the bone marrow and migrate into the periphery as mature cells. pDC development is directed by E2-2, a member of the E protein family of basic helix-loop-helix transcription factors that are obligate dimers and bind an E-box sequence. E proteins are antagonized by Id proteins, and Id family member Id2 is required for cDC development. The apparently cell intrinsic choice of pDC vs. cDC fate is determined by the net activity of E proteins. Loss of E2-2 affects only pDC development, but its expression is not as specific, as E2-2 is also expressed in cDCs, macrophages and B cells. E2-2 expression in cDCs was particularly puzzling, considering the dependence of cDCs on Id2, the E2-2 antagonist. We strived to address the discrepancy between the very specific activity of E2-2 in pDC development and its broader expression. This work shows that the E2-2 locus encodes two independently regulated isoforms. E2-2Long, the more active isoform is expressed only in pDCs, and E2-2Short, the less transcriptionally active isoform, is expressed more broadly. Moreover, E2-2Long is required for optimal pDC development. Homozygous loss of just E2-2Long leads to lower pDC frequency in the spleen and phenotypically affected pDCs in the periphery. Although E2-2 is essential for pDC development, no signal has yet been identified that induces E2-2 expression to influence pDC over cDC fate. Given the essential nature of E2-2 in pDC development, we aimed to understand better how E2-2 is regulated during pDC fate specification. We examined an E2-2Long reporter and found that E2-2Long expression precedes pDC commitment in early progenitors. E2-2 is only upregulated in committed pDCs, and actively shut down in cDCs through the expression of Id2. Analysis of E2-2 in an in vitro time controlled model of dendritic cell development showed that all dendritic cells (both cDCs and pDC) go through an E2-2 expressing stage only to resolve into E2-2- cDCs and E2-2+ pDCs. Early E2-2 expression and E2-2 upregulation upon pDC commitment hinted at the presence of an E2-2 controlled cis-regulatory module. ChIP-Seq data showed only one peak of E2-2 binding located 150 kb downstream of the TCF4 gene. Heterozygous deletion of this region in the in vitro DC development model led to impaired pDC development and a fail to undergo the E2-2+ stage described above. The regulatory element is essential for E2-2 upregulation during DC development. This work describes two new mechanisms of E protein regulation during dendritic cell development: cell specific differential isoform usage and a distal cis regulatory element responsible for enforcing and upregulating E2-2 expression. These new mechanisms can lead to better understanding of how this family of broadly expressed and pleiotropic transcription factors is regulated, with implications in overall development, not only dendritic cell fate decision.

Sequencing by Synthesis (SBS), a DNA sequencing methodology based on the DNA polymerase reaction, is a promising paradigm for deciphering large-scale genomes. This thesis describes the design and synthesis of a variety of nucleotide reversible terminators (NRTs) with different characteristics. One set of NRTs possesses a phosphate moiety attached to the 2’ position of the sugar to block further incorporation in polymerase reaction, with the potential for fluorescent tag attachment at the same site or on the base through a cleavable linker for detection. The other set of NRTs possesses an azido-methyl moiety that blocks the 3’-hydroxyl group for detection by surface-enhanced Raman scattering. Each NRT has been tested in proof-of-principle SBS experiments. In addition, a set of 5’-phosphate tagged nucleotides has been developed and tested for nanopore electronic detection. A new set of NRTs, 2’-O-monophosphate 3’-hydroxyl nucleoside 5’-triphosphates (2’-P-NTPs) has been synthesized and its application for SBS has been investigated (chapter 2). These NRTs contain a phosphate at the 2’ position of the sugar ring, which serves as the removable capping group during the polymerase reaction. This moiety is positioned close to the 3’-hydroxyl group so as to block further nucleotide incorporation in the polymerase reaction. It nonetheless should allow improved binding to the polymerase relative to nucleotides with blocking groups at the 3’ position, since polymerases have strict requirements for the 3’-OH binding pocket. 2’-P-NTPs can be incorporated into the growing nucleic acid strand at temperatures ranging from 37oC to 65oC with Stoffel fragment modified 19 (SfM19) polymerase. After incorporation, the phosphate capping moiety on the 2’ position of the DNA extension product can be efficiently removed by enzymatic phosphatase reaction permitting the next incorporation step. Fluorescently labeled 2’-P-NTPs have the potential for sequencing DNA and direct sequencing of RNA-like templates. As an alternative to fluorescence-based SBS, a Raman spectroscopy detection method was developed using an azido moiety (N3) as both a 3’-OH blocking group and a label with an intense, narrow and unique Raman shift at 2125 cm-1, where virtually all biological molecules are transparent (chapter 3). First the four 3’-O-azidomethyl nucleotide reversible terminators (N3-dNTPs) were demonstrated to produce surface enhanced Raman scattering (SERS) at 2125 cm-1. These 4 nucleotide analogues were used as substrates for the polymerase to perform a complete 4-step SBS reaction. SERS was used to monitor the appearance of the azide-specific Raman peak at 2125 cm-1 as a result of polymerase mediated primer extension by a single N3-dNTP and disappearance of this Raman peak upon cleavage of the azido label to permit the next nucleotide incorporation, thereby determining the DNA sequence. Due to the small size of the azido label, the N3-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for this moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS. Chapter 4 describes the design, synthesis and characterization of a new set of 5’-phosphate labeled nano-tag nucleotides (NTNs) for single molecule electronic SBS by nanopore detection. Four modified oligonucleotide polymers that produce distinct electrical current blockade signals in nanopores were designed as the nano-tags. While most of the NTNs flow rapidly through the pore, those complementary to the nucleotide on the DNA template are captured by the polymerase and will have at least 10-fold longer dwell times in the pore, which affords enough time for measuring and discriminating the signals. Since the nano-tags are automatically removed during the polymerase extension reaction in real time, only natural DNA strands are produced. Thus this SBS method should decrease the overall sequencing time and increase the read length.

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.

No description available.

Bibliography: leaves 121-139. The aim of this study was to determine the extent to which protein synthesis inhibition, lowered glutathione (GSH) levels and toxin metabolism contribute to the toxicity of cyclindrospermopsin. Both hepatocyte cultures and reticulocyte lysates were utilized as in vitro tools of investigation. The findings imply that the inhibition of protein synthesis by direct action of the toxin cannot be considered a primary cause of hepatocyte cell death over an acute time frame. Cytochrome P450-derived metabolites may play a crucial role in cytotoxicity, and the toxicity process does not appear to involve oxidative damage.

In recent years, the general public has recognized the dangers of a high fat diet and are demanding meat with lower fat content. This demand has stimulated research in the growth and regulation of adipocytes. However, despite much effort, no adipocyte-specific plasma membrane markers from any species are available as an aid to accurately distinguish adipocytes from non-adipocytes. One potential candidate for such a marker in porcine adipocytes has been identified by Killefer and Hu (1990b). Characterization of the cDNA for this protein, designated porcine adipocyte membrane protein (PAMP), is presented here. Sequence for the 910 by clone is 80% similar to an internal region of a rat prostaglandin F[subscript 2��] receptor regulator protein (FPRP) described by Orlickey (1996). Western blot analysis suggests that the pig protein is a homotetramer held together with disulfide bonds which form very close to the transmembrane region making the tetramer extremely difficult to reduce to monomeric units. Oligonucleotide primers were designed to amplify a genomic fragment by the polymerase chain reaction (PCR) and for a reverse transcriptase PCR (RT-PCR) assay to study the expression of the mRNA. A 2114 bp genomic clone revealed one intron in the coding region. A serum-free primary cell culture system was used to study the expression of the mRNA. Although message was detected every day over a ten day period, it appeared to peak between 6 to 8 days after plating. The PAMP protein is clearly of the same family as the rat FPRP but its size and conformation are quite different so it is not clear what function it performs in porcine adipocytes. Further experiments should focus on attaining full length cDNA's, confirming the molecular conformation of the protein, and assessing its function in a serum-free primary cell culture system. / Graduation date: 1997

Graduation date: 1995