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1
Bailey, Brenae L. "Stochastic Models of –1 Programmed Ribosomal Frameshifting." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/320007.
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Many viruses can produce multiple proteins from a single mRNA sequence by encoding the proteins in overlapping genes. One mechanism that causes the ribosomes of infected cells to decode both genes is –1 programmed ribosomal frameshifting. In this process, structural elements of the viral mRNA signal the ribosome to shift reading frames at a specific point. Although –1 frameshifting has been recognized since 1985, the mechanism is not well understood. I have developed a stochastic model of mRNA translation that includes the possibility of a –1 frameshift at any codon. The transition probabilities between states of the model are based on the energetics of local molecular interactions. The model reproduces observed translation rates as well as both the location and efficiency of frameshift events in the HIV-1 gag-pol sequence. In this work, the model is used to predict changes in the frameshift efficiency due to mutations in the viral mRNA sequence or variations in relative tRNA abundances. The model is sensitive to the size of the translating ribosome and to assumptions about the unfolding pathway of the stimulatory structure. As knowledge in the field of RNA structure prediction grows, that knowledge can be incorporated into the model developed here to make improved predictions. The single-ribosome translation model has been extended to polysomes by including initiation and termination rates and an exclusion principle, and allowing the stimulatory structure to refold on an appropriate timescale. The predicted frameshift efficiency for a given mRNA can be tuned by varying the ribosome density on the mRNA. This finding affects the interpretation of frameshift efficiencies measured in the lab. In the parameter regime where translation is initiation-limited, the frameshift efficiency also depends on the structure refolding rate, which determines the availability of the downstream structure for stimulating –1 frameshifts. Furthermore, there is a trade-off between frameshift efficiency and protein synthesis rate.
2
Vidaković, Marijana. "Studies of -1 ribosomal frameshifting in virus systems." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621823.
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Neeriemer, Jessica Joy. "Programmed ribosomal frameshifting in SARS-CoV and HIV-1." College Park, Md.: University of Maryland, 2007. http://hdl.handle.net/1903/7713.
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Thesis (M.S.) -- University of Maryland, College Park, 2007.Thesis research directed by: Dept. of Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
4
Nikolić, Emily Isabel Cinzia. "Structural and functional studies of programmed -1 ribosomal frameshifting." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607928.
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King, Louise Margaret. "Studies of programmed -1 ribosomal frameshifting in virus systems." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615622.
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Kontos, Charalampos. "A molecular analysis of the role of ribosomal pausing in -1 ribosomal frameshifting." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624652.
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Ramarao, Rachana. "Molecular studies of programmed -1 ribosomal frameshifting and translational readthrough." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615726.
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Liphardt, Jan Tage Carl. "The mechanism of -1 ribosomal frameshifting : experimental and theoretical analysis." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621577.
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Pennell, Simon John. "Structural studies of RNA pseudoknots involved in programmed -1 ribosomal frameshifting." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620323.
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Wang, Guan. "STRUCTURAL STUDY OF HUMAN CATENIN ΒETA-LIKE PROTEIN 1 AND DOUBLE RNA PSEUDOKNOTS IN -1 RIBOSOMAL FRAMESHIFTING." OpenSIUC, 2015. https://opensiuc.lib.siu.edu/dissertations/993.
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Project 1-- structure determination of human catenin-β-like protein 1 by x-ray crystallography Catenin-β-like protein 1 (CTNNBL1) is a highly conserved protein with multiple functions, one of which is to act as an interaction partner of the antibody-diversification enzyme activation-induced cytidine deaminase (AID) for its nuclear import and subnuclear trafficking. In this dissertation, the crystal structure of full-length human CTNNBL1 is reported. The protein contains six armadillo (ARM) repeats that pack into a superhelical ARM domain. This ARM domain is unique within the ARM protein family owing to the presence of several unusual structural features. Moreover, CTNNBL1 contains significant and novel non-ARM structures flanking both ends of the central ARM domain. A strong continuous hydrophobic core runs through the whole structure, indicating that the ARM and non-ARM structures fold together to form an integral structure. This structure defines a highly restrictive and discriminatory protein-binding groove that is not observed in other ARM proteins. The presence of a cluster of histidine residues in the groove implies a pH-sensitive histidine-mediated mechanism that may regulate protein binding activity. The many unique structural features of CTNNBL1 establish it as a distinct member of the ARM protein family. The structure provides critical insights into the molecular interactions between CTNNBL1 and its protein partners, especially AID. Project 2 -- study of double pseudoknots in the regulation of -1 programmed ribosomal frameshifting in RNA viruses −1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic and structural proteins at a defined ratio. For efficient −1 PRF, two cis-acting elements are required--a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. By searching for all possible pseudoknots within the full-length viral genomic mRNAs, we detected potential double pseudoknots at the −1 PRF junction in several animal viruses, including human immunodeficiency virus type-1 (HIV-1), transmissible gastroenteritis virus (TGEV), Barmah Forest virus (BFV), Fort Morgan virus (FMV), and Equine arteritis virus (EAV). We built structural models of the HIV-1 and EAV double pseudoknots to show that both the tandem and embedded mode of double pseudoknots are feasible and reasonable. We hypothesize that the fundamental reason for the viruses to utilize coaxially stacked double pseudoknots is to increase the overall stability of the frameshift regulating structure and while avoiding the use of an ultra stable single pseudoknot which may become a ribosomal roadblock. Results from this study significantly expand the repertoire of RNA structures and dynamics that may involve in the regulation of −1 PRF in viruses
11
Girnary, Roseanne Waheeda. "Structural and functional studies of the stimulatory RNAs involved in programmed -1 ribosomal frameshifting and translational readthrough." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612716.
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Caliskan, Neva [Verfasser], Marina [Akademischer Betreuer] Rodnina, Holger [Akademischer Betreuer] Stark, and Ralf [Akademischer Betreuer] Ficner. "Mechanisms of programmed ribosomal -1 frameshifting in bacteria / Neva Caliskan. Gutachter: Holger Stark ; Ralf Ficner. Betreuer: Marina Rodnina." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://d-nb.info/1054191476/34.
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Chamanian, Mastooreh. "A Novel HIV-1 Genomic RNA Packaging Element and its Role in Interplay between RNAPackaging and Gag-Pol Ribosomal Frameshifting." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1363902886.
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Korniy, Natalia [Verfasser], Marina [Akademischer Betreuer] Rodnina, Marina [Gutachter] Rodnina, and Holger [Gutachter] Stark. "Recoding of viral mRNAs by –1 programmed ribosome frameshifting / Natalia Korniy ; Gutachter: Marina Rodnina, Holger Stark ; Betreuer: Marina Rodnina." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1210702096/34.
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Caliskan, Neva. "Mechanisms of programmed ribosomal -1 frameshifting in bacteria." Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0022-5F19-2.
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Chung, Te-Pao, and 鍾得寶. "The Study of in vivo -1 Ribosomal Frameshifting Efficiency." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/33449619017685705270.
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碩士國立臺灣大學
分子與細胞生物學研究所
99
Two subunits (tau and gamma) of the DNA polymerase III holoenzyme in Escherichia coli are produced through efficient -1 programmed ribosomal frameshifting. The efficiency of frameshifting is determined by the elements around the shift site on mRNA, including the slippery sequence, secondary structure and the distance between them. In general, increasing the translation initiation frequency will shorten the space between neighboring ribosomes on the mRNA and then increase the possibility of polysome formation. As a result, once the secondary structure is unfolded by a preceding ribosome, it may remain open until the next ribosome reaches. Thus, the frameshifting efficiency may be decreased. In this study, we use the dnaX frameshifting motif as a model system to explore the relationship between polysome and frameshifting efficiency. Our results suggest that a downstream ribosome unfolds the secondary structure of the mRNA and blocks refolding when it is terminated on the stop codon just after the secondary structure. In the meantime, an upstream ribosome coming to the frameshifting site encounters no secondary structures. Thus, fewer ribosomes are induced to undergo -1 frameshifting. The frameshifting efficiency will be restored while the formation frequency of polysome decreases. Our results support that frameshifting efficiency may be affected not only by the sequence itself, but also by the interaction between ribosomes on the mRNA.
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Wang, Hao-Che, and 王顥哲. "The Study of Polyribosome Effect on -1 Ribosomal Frameshifting Efficiency." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/78162372232879942276.
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碩士國立臺灣大學
分子與細胞生物學研究所
102
In Esherichia coli, DNA polymerase III holoenzyme includes γ and τ subunits that are synthesized by regulation of the -1 frameshifting translation on mRNA of dnaX. Frameshifting efficiency can be determined by some elements on mRNA, which are the slippery sequence, secondary structure and Shine-Dalgarno sequence (SD sequence). Another element is polyribosomes, which occur at high frequency of initiation and cause the decrease of frameshifting efficiency. The reason that polyribosome decreases the frameshifting is the downstream ribosome blocks the refolding of secondary structures and prevents the upstream ribosome from interacting with the secondary structures. In our study, we use the dnaX frameshifting motif as a model system to study the influence of the density of polyribome to frameshifting efficiency in vivo and in vitro. In these studies, we control the induction time and concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for the in vivo experiments; in in vitro experiments, we change the translation time, the concentration of DNA plasmid or mRNA, and the fourth nucleotide of the stop codon. Our results show that with longer IPTG induction time or higher IPTG concentrations, the frameshifting efficiency was higher in in vivo experiment. For in vitro experiments, we observed that higher frameshifting efficiency comes from longer translation times or higher concentrations of the DNA plasmid or mRNA. Both results can be explained by the polyribosome effect; when mRNA concentration is increased, the ratio of ribosome to mRNA will be decreased, thus the density of polyribosome decreases to make frameshifting efficiency increased. Furthermore, the fourth nucleotide of the stop codon can influence termination efficiency; when the efficiency is too low, then the ribosome will pause longer to cause polyribosome effect and reduce frameshifting efficiency.
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Lin, Szu-Chieh, and 林思潔. "Regulation of -1 programmed ribosomal frameshifting by a metabolite-responsive RNA pseudoknot." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/52817314212305492649.
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碩士國立中興大學
生物化學研究所
98
There are many ways to regulate translation, and -1 programmed ribosomal frameshifting (-1 PRF) is one of them. -1 PRF is found in many type of viruses which can regulate the ratio of essential proteins by -1 PRF. Efficient -1 PRF requires two RNA elements. The first one is a hepta-nucleotide slippery sequence, located in the 5’ end named “slippery site”. The second element is a stimulator RNA structure, located 5-7 nucleotides downstream of the slippery site. There are many kinds of structures that can be a stimulator RNA, such as a simple hairpin in HIV-1, the H-type pseudoknot in BWYV or the three-stem pseudoknot in SARS-CoV. The most common of them is a hairpin-type pseudoknot. A specific RNA motif named 68 metH RNA was found in 2008. This RNA motif, as a riboswitch, can regulate S-adenosyl-L-methionine (SAM) recycling by binding S-adenosylhomocysteine (SAH). In addition, secondary structure prediction suggested that 68 metH RNA is a pseudoknot. Previously, we have confirmed the 68 metH RNA can induce -1 PRF in response to SAH concentration. I demonstrate here the influence of the base pairs in the 68 metH RNA on SAH-dependent -1 PRF by mutagenesis. When the stem regions was disrupted, no -1 PRF signal could be detected even in the presence of high SAH concentration. Therefore, the 68 metH RNA requires all the stem regions to induce SAH-dependent -1 PRF. Furthermore, I also demonstrate that increasing the stability of stem 2 can improve the SAH-dependent -1 PRF efficiency. Sequence alignment of SAH riboswitch suggests that several nucleotides are highly conserved. The 68 metH RNA could not induce -1 PRF when these highly conserved nucleotides were mutated. It thus implies that these nucleotides play important roles in SAH-dependent -1 PRF of 68 metH RNA.
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Chen, Cheng-Yu, and 陳政裕. "The functional study of -1 programmed ribosomal frameshifting attenuator in different cells." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/ncuz47.
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碩士國立中興大學
生物化學研究所
100
The translation processes in distinct organisms follow the genetic central dogma that messenger RNAs (mRNA) are transcribed from DNA, and proteins are translated from messenger RNA. However, translational process can be regulated by -1 programmed ribosomal frameshifting (-1 PRF) to produce particular fusion proteins, and is caused by the slippery sequence (XXXYYYZ) and stimulator RNA structure within mRNA sequences. We already demonstrated that the DU177 pesudoknot structure could stimulate -1PRF and an RNA hairpin 6BPGC can serve as a -1 PRF attenuator to reduce the -1 PRF efficiency when it appears on the upstream of the slippery site. In this work, I want to know if the function of the DU177 pesudoknot and 6BPGC hairpin are conserved in different cells. Previous methods in the study of -1 PRF efficiency are based on radioactive substance and dual luciferase assay. However they are either dangerous or inconvenient. Therefore, we want to establish a GST-Renilla luciferase (GST-RLuc) system which is safe and convenient, as an alternative method for the study of -1 PRF. We used the Shine-Dalgarno-like sequence (SD-like sequence) which had been known to regulate the -1 PRF to examine the usage of GST-RLuc system as an reliable reporter system. In my study, both the enzyme activity based assay of GST and the luminescence intensity of RLuc and the western blot reslults suggest that the GST-RLuc system is useful. Finally, I construct the DU177 pesudoknot structure and 6BPGC attenuator in GST-RLuc system, and espress them in different cells to analyze their roles in different cells.
20
Lin, Ya-Hui, and 林雅惠. "Regulation of -1 Programmed Ribosomal Frameshifting by Synthetic Riboswitch in Human Cells." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/61481004067063697123.
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博士國立中興大學
生物化學研究所
103
-1 programmed ribosomal frameshifting (-1 PRF) is a mechanism to regulate gene expression at the level of protein synthesis. -1 PRF occurs upon ribosomes decoding mRNA on a hepta-nucleotides slippery sequence (XXXYYYZ) followed by an optimally spaced stimulator pseudoknot. These signals on mRNA cause a fraction of ribosomes to shift into the -1 reading-frame. A co-translational refolding hairpin found in upstream of slippery sequence attenuates the stimulator pseudoknot-mediated -1 PRF efficiency, and was named “attenuator”. Riboswitch, structured RNA capable of binding cellular metabolites to control downstream gene expression by ligand-dependent RNA conformational change, has been characterized in the regulation of prokaryotic transcription termination and translation initiation. Because of the differences between prokaryotes and eukaryotes in transcription termination and translation initiation, it is difficult to apply riboswitch regulatory platform in eukaryotes. However, a SAH pseudoknot riboswitch found in prokaryotes has been demonstrated to stimulate -1 PRF after SAH binding. It provides an opportunity to apply riboswitch into eukaryote cells by -1 PRF gene expression platform. Furthermore, due to the similarity between co-translational attenuation hairpin and the co-transcriptional termination hairpin in prokaryotic rho-independent transcription termination, I rationalized that -1 PRF attenuation event might also be regulated by a synthetic riboswitch. Here, I engineered a theophylline aptamer within the attenuator hairpin and demonstrated that the theophylline-responsive attenuators regulate -1 PRF by modulating the attenuator hairpin formation or disruption. The combination of the upstream theophylline-responsive attenuator and downstream SAH pseudoknot stimulator has synergistic effect on -1 PRF regulation. However, SAH pseudoknot stimulator causes the regulation leakages as SAH is a universal metabolite in cells. To overcome this problem, I engineered a non-cellular metabolite- theophylline aptamer into a stimulator pseudoknot to regulate -1 PRF by theophylline-dependent stimulator pseudoknot conformational rearrangements. Coupling of both theophylline-responsive upstream attenuator hairpin and downstream stimulator pseudoknot leads to better dynamic range on -1 PRF regulation. This synthetic riboswitch based -1 PRF gene expression extends the riboswitch application to a new gene expression platform and increases the gene regulation repertoire in mammalian cells.
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Kuo, Tai-Chin, and 郭黛瑾. "Regulation of -1 programmed ribosomal frameshifting in trans by small single-stranded RNA." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/90612600237412879376.
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碩士國立中興大學
生物化學研究所
98
The mechanism for the induction of −1 programmed ribosomal frameshifting(-1 PRF) requires two major elements: a heptanucleotide slippery site and a downstream RNA pseudoknot structure connected by a spacer. It has been demonstrated that RNA pseudoknot, hTPK-DU177 (DU177), which is derived from human telomerase RNA, can induce the -1 PRF efficiency of 50%. When the specific base-triple of DU177 was mutated to disrupt the interaction of base-triple, the efficiency of -1 PRF was seriously impaired even if the DU177 still adopts a pseudoknot conformation. Therefore, the base-triple interaction is essential for DU177 to induce -1 PRF. When DU177 was separated into two portions with one portion constructed in reporter mRNA and the other as a single-stranded RNA, to mimic DU177 via interaction of base-triple, it can stimulate -1 PRF in trans. The -1 PRF induced by an in-trans RNA via specific base-triple interaction with mRNA can thus be a plausible function for non-coding RNAs. My works focus on two goals: (1) enhance the -1 PRF efficiency stimulated by the DU177 lacking a loop-closure feature (loop 2). This is achieved by mutating the AU base-pairs, which will not affect the -1 PRF efficiency induced by the intact DU177, to GC base-pairs. These GC base-pairs can strengthen the interaction between a reporter mRNA and its corresponding in-trans RNA and thus enhance the efficiency of -1 PRF in-trans. The data showed that, GC base-pairs may improve the -1 PRF efficiency of DU177 lacking intact loop 2. (2) DU177 was separated with cut in loop 1 into two portions, a single(93-106 single) and a hairpin(107-184 hairpin). I then inserted the single or the hairpin into the reporter gene, while using the other one as the corresponding in-trans RNA to examine the -1 PRF induced in-trans. The data showed that the reporter gene mRNA containing 93-106 single can induce slightly -1 PRF efficiency. However, the other mRNA containing 107-184 hairpin can’t stimulate -1 PRF. As the mRNA 93-106 single’s corresponding in-trans RNA is a hairpin and thus be more stable, it can be applied to study in vivo -1 PRF regulation in the future.
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Yang, Zih-Liang, and 楊子諒. "Regulation of -1 Programmed Ribosomal Frameshifting by an S-adenosylmethionine-responsive RNA pseudoknot." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/48225291165311531880.
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碩士國立中興大學
生物化學研究所
104
During translation, ribosome is responsible for reading-frame maintenance to guarantee codons translated into amino acids in correct order and folded into functional proteins. However, some specific signals within mRNAs can induce a fraction of elongating ribosomes to move one base backward in the 5’ direction, and resume elongating on the -1 reading frame, which would produce different protein product from the 0-frame encoded protein. This event is called -1 programmed ribosomal frameshifting (-1 PRF). An efficient -1 PRF signal within mRNAs requires two elements: a heptanucleotide slippery sequence (XXXYYYZ) and a downstream RNA stimulator, usually a hairpin-typed pseudoknot. Riboswitches, structured RNAs, can bind specific metabolites and control gene expression by metabolites-dependent structural change. In 2010, our lab found a pseudoknot derived from S-adenosylhomocysteine (SAH) riboswitch can trigger SAH-dependent -1 PRF in mammalian cells. SAH is the product from S-adenosylmethionine (SAM) mediated methylation reactions with the methyl group of SAM being transferred to particular targets, such as DNA, RNA or protein. Several SAM riboswitches have been found in bacteria, possessing different complicated RNA structures for sensing and binding SAM. Interestingly, some of them are pseudoknot. Thus, these SAM-sensing pseudoknots might have the ability to stimulate -1 PRF occurred. In this study, I found and demonstrated a pseudoknot derived from SAM-II riboswitch (SAM-II Bth) could induce SAM-dependent -1 PRF. To further improve its -1 PRF efficiency, I made some mutations in SAM-II Bth pseudoknot to increase the stability of stem regions of the pseudoknot. However, this improved SAM-II Bth pseudoknot only stimulate -1 PRF efficiency ~1 % in mammalian cells. This SAM-sensing pseudoknot will need further improvements for practical applications.
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Chang, Hui-Ting, and 張惠婷. "Effect of spacer-length on -1 programmed ribosomal frameshifting of different stimulator RNAs." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/53165089815237730925.
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碩士國立中興大學
生物化學研究所
103
Ribosome decodes the reading frame of messenger RNA (mRNA) to synthesize protein during translation. However, the mRNAs contain the signasl that are sufficient to cause -1 programmed ribosomal frameshifting (-1PRF). The signals include a hepta-nucleotide slippery site, a stimulator RNA structure, and a proper spacer distance. According to the -1PRF model, frameshifting could occur during the accommodation of aminoacyl-tRNA into the A-site or translocation stage of a translation elongation cycle. When the elongating ribosome encounters the stable and complicated stimulator RNA, the attempt to unwind the stimulator provides additional torsional resistance to ribosome movement, and cause the elongating ribosome to pause at the slippery site. Therefore, the stimulator RNA blockage at the entrance tunnel caused the ribosome to build up tension in the spacer region. The tension is released via disrupting the codon-anticodon interaction at the slippery site allowing the movement of tRNA to slip toward -1 direction. Thus, frameshifting efficiency could be correlated to the tension force built up in the spacer region. Previous study reveals that the correct spacing distance must be maintained between the slippery sequence and the stimulator RNA, and changing the spacer distance could affect -1 frameshifting efficiency (Brierley et al., 1989). My research aims to investigate the influence of spacer-length on -1 PRF of different stimulator RNAs. The -1 PRF constructs contain a range of spacer lengths, and then inserted into the reporter gene to measure -1 frameshifting efficiency. Here, we show the -1 frameshifting efficiency stimulated by the DU177 pseudoknot is more sensitive than those of SRV-1 and JEV pseudoknots in response to the variation of spacer lengths. In addition, the optimal spacer-length for efficient -1PRF activity stimulated by DU177 pseudoknot is different between the two different eukaryotic ribosomes tested, and may by caused by difference in the ribosome sizes. Furthermore, we constructed an RNA hairpin upstream of the slippery sequence to function as -1 frameshifting attenuator for the DU177 pseudoknot, and found that -1PRF attenuation efficiency was also affected by the spacer-length. Together, these results indicate that the effect of spacer-length in -1 frameshifting efficiency is also affected by the structural features of the stimulator used.
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Chou, Ming-Yuan, and 周銘源. "The important structural features of the DU177 RNA pseudoknot in stimulating programmed -1 ribosomal frameshifting." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/58425338942445395540.
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博士國立中興大學
生物化學研究所
98
Programmed -1 ribosomal frameshifting (-1 PRF), which is known to be caused by the slippery sequence and the stimulator sequence, modulates the moving direction and distance of a translating ribosome. Upon reaching the slippery sequence, the translation complex tends to shift into the -1 reading frame. However, the tRNAs in the translation complex can still form stable codon-anticodon interactions after shifting into the -1 frame because of the composition of the slippery sequence. The stimulator sequence, properly distant from the slippery sequence, is regarded to be the main source to pause the translation complex on the slippery sequence and cause -1 PRF, and thus the lever that controls -1 PRF efficiency. We found DU177, an RNA pseudoknot derived from the human telomerase RNA, can stimulate -1 PRF. With its detailed structural data, we tried to understand the important structural features of the DU177 pseudoknot responsible for its -1 PRF efficiency through mutagenesis. Our results indicated both the interactions between stem and loop and the stacking junction of the two stem helixes have greater effect on the -1 PRF stimulating property of the DU177 pseudoknot than the loop nucleotides near the RNA entry channel of the ribosome when -1 PRF occurred. We divided the DU177 pseudoknot into a hairpin structure on mRNA and an oligo RNA, and we found -1 PRF occurred after the stem loop interactions applying on the hairpin structure. In addition to confirming the importance of stem loop interactions and junction stacking in -1 PRF, we also found an single-strand RNA inducible -1 PRF stimulator. The inducible -1 PRF stimulator was converted to stimulate -1 PRF after the binding of its responsive molecule and is suitable for application in the study of -1 PRF mechanism. Because of the correlation between translation elongation mechanism and -1 PRF, the study might help in understanding the details of translation translocation. Besides, the inducible -1 PRF stimulator might directly be applied on living organisms and has the potential to become an inducible translation control system in bioassay and medicine.
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Korniy, Natalia. "Recoding of viral mRNAs by –1 programmed ribosome frameshifting." Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C124-A.
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Stochmanski, Shawn Joseph. "Biology and characterisation of polyalanine as an emerging pathological marker." Thèse, 2014. http://hdl.handle.net/1866/13553.
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Dix-huit maladies humaines graves ont jusqu'ici été associées avec des expansions de trinucléotides répétés (TNR) codant soit pour des polyalanines (codées par des codons GCN répétés) soit pour des polyglutamines (codées par des codons CAG répétés) dans des protéines spécifiques. Parmi eux, la dystrophie musculaire oculopharyngée (DMOP), l’Ataxie spinocérébelleuse de type 3 (SCA3) et la maladie de Huntington (MH) sont des troubles à transmission autosomale dominante et à apparition tardive, caractérisés par la présence d'inclusions intranucléaires (IIN). Nous avons déjà identifié la mutation responsable de la DMOP comme étant une petite expansion (2 à 7 répétitions supplémentaires) du codon GCG répété du gène PABPN1. En outre, nous-mêmes ainsi que d’autres chercheurs avons identifié la présence d’événements de décalage du cadre de lecture ribosomique de -1 au niveau des codons répétés CAG des gènes ATXN3 (SCA3) et HTT (MH), entraînant ainsi la traduction de codons répétés hybrides CAG/GCA et la production d'un peptide contenant des polyalanines. Or, les données observées dans la DMOP suggèrent que la toxicité induite par les polyalanines est très sensible à leur quantité et leur longueur.
Pour valider notre hypothèse de décalage du cadre de lecture dans le gène ATXN3 dans des modèles animaux, nous avons essayé de reproduire nos constatations chez la drosophile et dans des neurones de mammifères. Nos résultats montrent que l'expression transgénique de codons répétés CAG élargis dans l’ADNc de ATXN3 conduit aux événements de décalage du cadre de lecture -1, et que ces événements sont néfastes. À l'inverse, l'expression transgénique de codons répétés CAA (codant pour les polyglutamines) élargis dans l’ADNc de ATXN3 ne conduit pas aux événements de décalage du cadre de lecture -1, et n’est pas toxique. Par ailleurs, l’ARNm des codons répétés CAG élargis dans ATXN3 ne contribue pas à la toxicité observée dans nos modèles. Ces observations indiquent que l’expansion de polyglutamines dans nos modèles drosophile et de neurones de mammifères pour SCA3 ne suffit pas au développement d'un phénotype.
Par conséquent, nous proposons que le décalage du cadre de lecture ribosomique -1 contribue à la toxicité associée aux répétitions CAG dans le gène ATXN3.
Pour étudier le décalage du cadre de lecture -1 dans les maladies à expansion de trinucléotides CAG en général, nous avons voulu créer un anticorps capable de détecter le produit présentant ce décalage. Nous rapportons ici la caractérisation d’un anticorps polyclonal qui reconnaît sélectivement les expansions pathologiques de polyalanines dans la protéine PABPN1 impliquée dans la DMOP. En outre, notre anticorps détecte également la présence de protéines contenant des alanines dans les inclusions intranucléaires (IIN) des échantillons de patients SCA3 et MD.Eighteen severe human diseases have thus far been associated with trinucleotide repeat (TNR) expansions coding for either polyalanine (encoded by a GCN repeat tract) or polyglutamine (encoded by a CAG repeat tract) in specific proteins. Among them, oculopharyngeal muscular dystrophy (OPMD), spinocerebellar ataxia type-3 (SCA3), and Huntington’s disease (HD) are late-onset autosomal-dominant disorders characterised by the presence of intranuclear inclusions (INIs). We have previously identified the OPMD causative mutation as a small expansion (2 to 7) of a GCG repeat tract in the PABPN1 gene. In addition, we and others have reported the occurrence of -1 ribosomal frameshifting events in expanded CAG repeat tracts in the ATXN3 (SCA3) and HTT (HD) genes, which result in the translation of a hybrid CAG/GCA repeat tract and the production of a polyalanine-containing peptide. Data from OPMD suggests that polyalanine-induced toxicity is very sensitive to the dosage and length of the alanine stretch. To validate our ATXN3 -1 frameshifting hypothesis in animal models, we set out to reproduce our findings in Drosophila and mammalian neurons. Our results show that the transgenic expression of expanded CAG repeat tract ATXN3 cDNA led to -1 frameshifting events, and that these events are deleterious. Conversely, the expression of polyglutamine-encoding expanded CAA repeat tract ATXN3 cDNA was neither frameshifted nor toxic. Furthermore, expanded CAG repeat tract ATXN3 mRNA does not contribute to the toxicity observed in our models. These observations indicate that expanded polyglutamine repeats in Drosophila and mammalian neuron models of SCA3 are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with CAG repeat tract expansions in the ATXN3 gene. To further investigate ribosomal frameshifting in expanded CAG repeat tract diseases, we sought to create an antibody capable of detecting the frameshifted product. Here we report the characterization of a polyclonal antibody that selectively recognizes pathological expansions of polyalanine in the protein implicated in OPMD, PABPN1. Furthermore, our antibody also detects the presence of alanine proteins in the intranuclear inclusions (INIs) of SCA3 and HD patient samples.