Dissertations / Theses on the topic 'Ribosome binding sites'

Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator. A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression.
Master of Science
Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.

Investigators have been attempting to identify the receptor for ribosomes on the rough endoplasmic reticulum (RER) for almost 20 years, yet the ribosome receptor has remained elusive. Rough microsomal membranes contain endogenous ribosomes bound in at least two types of interactions. Loosely associated ribosomes can be removed by extraction with a high ionic strength solution, but ribosomes that were actively engaged in translocation when the membranes were isolated remain tethered to the membrane by a nascent polypeptide (Adelman et al., 1973). The original assay for the ribosome receptor involved stripping all of the endogenous ribosomes off of intact membranes before adding back a quantitated amount of ribosomes. More recent assays have employed detergent solubilization of the membrane and then reconstitution of the membrane proteins into lipid vesicles before adding back ribosomes. In both cases ribosome binding to its receptor is measured in an assay that does not involve translation or translocation. We utilized a crosslinking assay to attempt to identify membrane proteins that function as a binding site for ribosomes engaged in protein translocation across the endoplasmic reticulum. In vivo bound ribosomes that remain associated with the membrane after extraction with a high ionic strength solution are likely to be bound to a functional translocation site. The water soluble, membrane impermeable, thiol-cleavable crosslinker 3,3'-dithiobis (sulfosuccinimidylpropionate) was selected to limit reaction to protein domains located on the cytoplasmic face of salt extracted microsomal membrane vesicles. A specific subset of RER proteins was reproducibly crosslinked to the endogenous ribosomes. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35 kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180 kD protein that had recently been proposed to be a ribosome receptor (Savitz and Meyer, 1990). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes in an in vitro binding assay to determine whether ribosome binding activity could be ascribed to the 180 kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180 kD protein, as determined by Coomassie blue staining of a polyacrylamide gel. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments and functional assays with proteoliposomes demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180 kD protein by chromatography. Taken together, the evidence indicates that the 180 kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor. To continue the investigation of ribosome binding, an assay was designed to characterize ribosome-nascent chain complexes bound to the microsomal membrane during translocation. A series of translocation intermediates consisting of discrete sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. Proteinase K was then used as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport. Only those partially translocated nascent chains of 100 amino acids or less were insensitive to protease digestion by externally added protease. It was concluded that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, shorter nascent polypeptides appear not to have lumenally exposed segments. Ultimately, a functional assay for the ribosome receptor should include binding studies conducted under physiological conditions. For this purpose, an assay was developed that allowed translation, translocation, and termination of a secretory protein to be monitored with probes designed to independently quantitate translating and non-translating ribosomes. A synchronized wheat-germ translation system was programmed with bovine preprolactin mRNA and aliquots were taken at various time points before and after adding membranes. The samples were then separated into membrane bound and soluble species by centrifugation. RNA was isolated from each supernatant and pellet sample and blotted onto nylon sheets. By probing the dot blots with probes that hybridize with either the 5S RNA of wheat germ ribosomes or the preprolactin transcript, the translating ribosomes could be monitored without the interference of the endogenous canine ribosomes on the membrane. By comparing the total amount of preprolactin transcript that bound to the membrane versus the total amount of wheat germ ribosomes bound to the membrane, it was discovered that the vast majority (>99%) of wheat germ ribosomes that bound to the microsomal membrane were non-translating ribosomes. In later experiments it was found that the non-translating ribosomes did not compete with the translating ribosomes; under all conditions tested, the translating ribosomes had access to translocation sites on the microsomal membrane. One interpretation of this data is that all ribosome binding sites are not identical. It may be that functional sites for translocation are a distinct subclass of total ribosome binding sites. Another interpretation is that a ribosome in a nascent chain-SRP complex has a much higher affinity for the ribosome receptor than nontranslating ribosomes or 60S subunits. Perhaps the non-translating ribosome can not compete with ribosomes engaged in translocation. As stated earlier, ribosomes do make at least two kinds of interactions with the microsomal membrane surface. This data may be indicative of those types of interactions.

Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme's substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.
Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding.
Revisión por pares

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1998.
Includes bibliographical references (p. 342-353).
The yeast (Saccharomyces cerevisiae) ribosomal protein L30 and its auto-regulatory pre-mRNA binding site provide one of the best examples the critical role of protein-RNA interactions in regulation of RNA processing and control of gene translation. A model system for this interaction, which includes the ribosomal L30 protein and the phylogenetically conserved RNA segment for auto-regulation, was studied using nuclear magnetic resonance (NMR) spectroscopy. The L30 protein recognizes and binds tightly to the stem-internal loop-stem RNA, the recognition elements of which lie mostly on the conserved two-plus-five asymmetric purine-rich internal loop. NMR characterizations were carried out on both the free and bound forms of the protein and the RNA. Detailed analyses of the protein revealed that the main architecture, a fourstranded n-sheet sandwiched between four a-helices, is present both in the free and in the bound form. There are however, substantial local perturbations that accompany RNA binding, the largest of which have been mapped onto the loops connecting Strand A and Helix 2, Strand B and Helix 3, Helix 4 and Strand D. In contrast to the protein, the internal loop of the RNA undergoes significant changes upon complex formation, and the most distinct observation was the formation of the G 11G56 reverse Hoogsteen mismatch pair. Structure modeling using simulated annealing in restrained molecular dynamics was carried out in X-PLOR. Detailed analyses of the complex structure reveal that the protein recognizes the RNA mostly along one side of the internal loop with five purines. The interactions are divided further into two sections. One region consists of mostly aromatic stacking and hydrophobic contacts from Leu25, Phe85 and Val87 of the protein to G56 of the RNA. The other region consists of mostly specific contacts, which include recognition of A57 by Asn 48, and G58 by Arg 52. The L30 protein- RNA complex structure thus determined using NMR spectroscopy not only provides a detailed insight for understanding the structure-function relationship regarding the yeast auto-regulation, it also further demonstrates the important role of the protein-RNA interaction in controlling RNA processing and gene translation.
by Hongyuan Mao.
Ph.D.

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack. In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools. Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods. Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.

Biologically synthesized poly(α,L-glutamic acid) (PLGA) can be chemically modified to form monodisperse poly(γ-benzyl α,L-glutamate) (PBLG). This material shows rare smectic ordering where macromolecular rods organize into highly-ordered layers. Analysis of PBLG has been hindered by low level biosynthesis of PLGA. An unusual feature of PLGA expression is that its accumulation is inversely related to the levels of its mRNA. This phenomenon has been investigated with the objective of improving the bioproduction of PLGA. PLGA was expressed as a C-terminal fusion with dihydrofolate reductase (DHFR) in E. coli strain BL21, carrying an IPTG-inducible DHFR-PLGA gene fusion in the pQE15 plasmid. In exponentially growing cells, accumulation of DHFR-PLGA was optimal at 0.01 mM IPTG and decreased at higher concentrations of IPTG. However, maximal DHFR-PLGA accumulation occurred in cells grown to saturation with no IPTG induction. It appears, therefore, that the accumulation of DHFR-PLGA is optimal in nongrowing cells translating low levels of DHFR-PLGA mRNA over long periods of incubation. Overexpression of both E. coli tRNAGlu and the glutamyl-tRNA synthetase did not improve DHFR-PLGA production. In vivo incorporation of [35S]-methionine was inhibited >95% by induction of either translatable or untranslatable PLGA constructs, but induction of the corresponding anti-sense constructs was not inhibitory. Sucrose gradient centrifugation analysis showed that expression of PLGA RNA resulted in nearly complete depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. These new complexes were enriched in 16S rRNA but also contained 23S rRNA, and unlike normal polysomes, they were resistant to breakdown in the presence of puromycin. These results support the conclusion that multiple internal ribosome binding sites in the PLGA coding sequence inhibit translation of both DHFR-PLGA and cellular proteins by sequestering ribosomal subunits in nonfunctional complexes on the PLGA mRNA.

碩士
長庚大學
醫學生物技術研究所
97
The 5’ untranslation region (5’UTR) of enterovirus 71 (EV71) contains internal ribosome entry site (IRES) that directs the initiation of viral protein translation. In addition to viral IRES, numerous cellular mRNAs , such as cellular human immunoglobulin heavy chain-binding protein (BiP), c-myc and cyclin D1 have been reported to contain IRES . Furthermore, many cellular IRES have also been found to be activated upon stress, such as virus infection. Several IRES trans-acting factors (ITAFs) for EV71 have been identified in our previous study, including far upstream element (FUSE) binding protein 1 (FBP1) and far upstream element (FUSE) binding protein 2 (FBP2). Some ITAFs of EV71 also can interact with cellular IRES. The study attempts to examine the activation of cellular IRES during EV71 infection and to investigate the roles of FBP1 and FBP2 on viral IRES versus cellular IRES. The protein expression level of BiP increased slightly upon EV71 infection. Both the IRES activity of EV71 and BiP were increase in EV71-infected cells. The interactions of FBP1 and FBP2 with BiP were confirmed by RNA pull down assay and competition assay. The IRES activity of EV71 and BiP decreased in FBP1 siRNA-treated cells. However, EV71 IRES activity increased in FBP2-knockdown cells, in contrast to IRES activity of BiP in FBP2-knockdown cells. The results suggest that FBP1 may act as a positive regulator of EV71 and BiP IRES. However, FBP2 is a negative regulator of EV71 IRES, but it is a positive regulator of BiP IRES.

Thesis (Ph. D.)--University of Notre Dame, 2005.
Thesis directed by Paul W. Huber for the Department of Chemistry and Biochemistry. "November 2005." Includes bibliographical references (leaves 120-130).

Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG http://corg.molgen.mpg.de, a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server http://tomcat.molgen.mpg.de:8080/das. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Interaction of ribosomal protein L1 and the 23S rRNA plays an important role in both the structure and biological activity of the Escherichia coli ribosome. We have minimized the binding site for protein L1 on the 23S rRNA to a 32-nucleotide fragment consisting of helix 77, helix 78, and the conserved sequences that interconnect them. Filter-binding, modification-interference and manganese rescue experiments were used do demonstrate (1) that the absence of a 2′-OH group at position 2122 can disrupt protein L1-23S rRNA interaction, but only if certain other deoxyribonucleotides are present in the transcript and (2) that the Rp phosphoryl oxygens preceding U2122 and A2176 in the rRNA molecule play a role in Ll-23S rRNA interaction through magnesium ion coordination, possibly by participating in magnesium bridges with the protein. The crystal structure of protein L1 from Thermus thermophilus, which closely resembles Escherichia coli L1, consists of two domains that are divided by a deep cleft. It has been suggested that the binding site for RNA is located within this interdomain cleft. Using site-directed mutagenesis it was possible to identify a cluster of conserved amino acids that are crucial for protein-RNA interactions (F37, D42, T216, G218), as well as several amino acids that help to stabilize the complex (K36, P137, N138, K140, H171, K176, M217). All of the mutagenized amino acids are located on the surface of the interdomain cleft. To orient L1 relative to the RNA, crosslinking studies were performed using photoreactive moieties incorporated into the L1 binding site and protein L1 itself. A 4-thiouridine residue at position 2172 and 2-azidoadenosine ligated to the 3′-end of the minimized binding site were shown to form a “zero length” crosslink with protein L1. Whereas the azidophenacyl group attached to position 40 of the protein was demonstrated to map a portion of loop 76/77 on 23S rRNA.

博士
國立清華大學
分子醫學研究所
98
Molecular dynamics (MD) simulations allow detail analysis of structural dynamics of atomic–level phenomena such as binding recognition fundamental in Biology field. Binding interaction involved between (bio) –molecules can be evaluated by binding free energy calculation base on the law of thermodynamics. Conformational flexibility essential for investigating dynamic property can be estimated by calculating conformational entropy such as principal components analysis. Combination with these techniques can provide reasonable explanations for atomic–level phenomena that are difficult to explain on the basis of static models alone. Here we present the results of a series of conventional MD simulations on recognition and interaction between (I) a mithramycin dimer and a DNA duplex, (II) several aminoglycoside antibiotics and an oligonucleotide corresponding to rRNA A–site. Both kinds of antibiotics consist of a core structure where several sugar ring substitutions at different carbon positions. In part I of the study, we successfully built the dynamics model corresponding to the experimental structure and binding affinity, discussed the binding interaction, and found the cooperativity between this GC–specific DNA binding antibiotic and a decanucleotide duplex of two GC binding sites to be in an anticooperative manner. Following the MD protocol and modification of the force field parameters for this sugar–linked antibiotic, in part II of the study, we compared the binding recognition and hydration patterns between several aminoglycoside antibiotics and a RNA duplex corresponding to the aminoacyl–tRNA decoding site (A–site) of the 16S rRNA on the 30S subunit which is a crucial component of the bacterial translational machinery. We have built several dynamic models with reasonable binding free energies showing good linear correlation with the experimental data. The hydration sites around the U1406·U1495 pair in the A–site were analyzed to distinguish tightly bound water molecules from fast–exchanging ones which has been suggested to be useful for rational drug design. We found that the hydration sites with long residence time identified between ring III of two 4,6–disubstituted antibiotics (tobramycin and kanamycin A) and phosphate oxygen atoms of G1405/U1406 may be worthy of further exploration for rational design of this kind.

Cellular response to various stress conditions involves regulation of gene expression by different mechanisms. Translation is the final step in the flow of genetic information and regulation at this level allows an early response to changes in physiological conditions. Initiation of translation is the rate-limiting step of protein synthesis and hence is tightly regulated. Translation initiation in mammalian cells is mainly by “cap dependent pathway” wherein the 5’methyl guanosine “cap” structure is recognized by certain canonical initiation factors along with 40S ribosomal subunit and the complex scans the 5’UTR till it recognizes initiator AUG. This leads to the joining of the 60S ribosomal subunit and the initiation of translation. In an alternate mode of translation initiation called as the Internal ribosome entry site mediated translation (IRES), the ribosomes are recruited closer to the initiator AUG in a 5’ cap independent manner. Efficient translation by IRES mode requires some canonical initiation factors like eIF2 and eIF3 and other non-canonical IRES-trans-acting factors (ITAFs), which include human La antigen, polypyrimidine-tract binding protein (PTB),Upstream of N-Ras (Unr), Poly (rC) binding protein (PCBP2) etc. Various types of stress conditions, such as starvation of growth factors, heat shock, hypoxia, viral infection lead to down regulation of protein synthesis. However, translation of a subset of mRNAs continues or is up-regulated. Many of these mRNA may be translated by an IRES mode. It is believed that cellular IRESs become active during such conditions that abrogate the cap-dependent mode of translation so that the pool of vital proteins is maintained in the cell. In this thesis, presence of ‘IRES’ element has been investigated in the 5’UTR of Interferon regulatory factor -2 (IRF2) mRNA and the possible physiological significance has been studied. Further, it has been shown that polypyrimidine tract binding protein or PTB is important for the IRES activity. The probable mechanism of action of PTB has also been investigated which suggests that PTB interaction alters the IRF2 IRES conformation thus facilitating translation initiation. In the first part of the thesis, mRNAs that continue to be translated under heat-shocked condition, which is known to abrogate cap-dependent translation initiation, has been investigated by cDNA micro-array hybridization analysis of the ribosome bound RNA. The global protein synthesis was severely impaired under heat shock; however a number of mRNAs continued translation under this condition. Some of these mRNAs encode proteins that are likely to be involved in the heat shock response. Few of these genes are also reported to contain IRES element. Since the micro-array was performed from the RNA extracted from ribosome bound mRNA fraction in a condition when cap-dependent translation is impaired, it was hypothesized that some of the genes, which are up regulated under such condition, might operate via cap-independent mode of translation initiation. Based on this study, one candidate gene, the ‘interferon regulatory factor 2 (IRF2)’ was selected from the pool of up regulated genes and presence of an IRES element was investigated. Interferon regulatory factors are DNA-binding proteins that control interferon (IFN) gene expression. IRF2 has been shown to function as repressor of IFN and IFN-inducible genes. Real–Time and semi-quantitative RT-PCR assays were performed which validated the micro-array data. In the second part of the thesis, the presence of IRES element in the 5’UTR of IRF2 was investigated. Bicistronic assay showed comparable IRES activity with a known representative IRES, BiP, thus suggesting the presence of an IRES element in the IRF2 5’UTR. Stringent assays were then performed to rule out cryptic promoter activity, re-initiation/scanning or alternative splicing in the 5’UTR of the IRF2. RNA transfections using in vitro synthesized bicistronic RNAs further validated the presence of the IRES element. To understand the physiological significance of an IRES element in IRF2 mRNA, the cells were subjected to various stress conditions and IRES activity was studied. It seems IRF2 IRES function might not be sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease, which is known to cleave eIF 4G and thus inhibit the cap-dependent translation. Incidentally, Hepatitis A virus IRES was affected under such condition. Additionally, it was observed that compared to HCV or Bip IRES, the effect of Interferon α treatment was not so pronounced on the IRF2 IRES. This was further evidenced by its unchanged protein level post-treatment with interferon α. Furthermore, in cells treated with tunicamycin (a known agent causing ER stress), the IRF2 IRES activity and the protein levels were unaffected, although the cap dependent translation was severely impaired. The observations so far suggested that the IRF2 protein level is practically unchanged under conditions of ER stress and interferon treatment. Metabolic labeling followed by immunoprecipitation of IRF2 in cells treated with either tunicamycin or interferon suggested that de novo synthesis of the protein is continued under the above conditions thus validating our earlier data. In the third part of the thesis, the role of an IRES trans acting factor, PTB, in modulating the IRF2 IRES activity has been investigated. Analysis of the cellular protein binding with the IRF2 IRES suggested that certain cellular factors might influence its function under stress conditions. The IRF2 IRES was found to interact with a known trans-acting factor or PTB. To study the possible role of this trans acting factor, the PTB gene was partially silenced by PTB specific siRNA. This led to a decrease in the IRF2 IRES activity, suggesting that PTB is probably essential for the IRES activity. Interestingly, when Hela cells (with partially silenced PTB) were treated with tunicamycin (inducer of ER stress) the level of IRF2 protein was also found to be less thus pointing to an important role of PTB in IRF2 protein synthesis under such conditions. Western blot analysis and immunofluoroscence assay suggested that there was no significant nuclear-cytoplasmic relocalization of PTB under the condition studied. Primer extension inhibition assay or Toe-printing analysis was performed to detect the contact points of PTB on the IRF2 5’UTR. Many toe-prints were found on the 3’ end of the 5’UTR RNA. A 3’ deletion mutant was generated that showed reduced PTB binding. Incidentally the IRES activity of the mutant was also found to be less than the wt IRF2 RNA. Subsequently, structural analysis of the RNA was performed using enzymatic (CV1, RNase T1) and chemical modification (DMS) agents. Footprinting assay in presence of PTB suggested that there is change in the structure when PTB interacts with the RNA. To investigate this further, CD spectrum analysis of the IRF2 RNA in the presence of PTB was performed which indicated that there was a conformational change under such condition thus validating our earlier observation. The thesis reveals a novel cellular IRES element in the 5’UTR of IRF2 mRNA. The characterization of the IRES and possible role played by PTB protein in modulating its activity suggests that the regulated expression of IRF2 protein by its IRES element under various stress conditions would have major implications on the cellular response. Incidentally, this study constitutes the first report on translational control of interferon regulatory factors by internal initiation. The results might have far reaching implications on the possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general.

Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.