Functional Analysis of the RNA Binding Domains of the E. Coli NUSA Protein PDF Download
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Author: Ying Zhou
Publisher:
ISBN:
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Languages : en
Pages : 396
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Author: Ying Zhou
Publisher:
ISBN:
Category :
Languages : en
Pages : 396
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Author: Thien-Fah Mah
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Category :
Languages : en
Pages : 0
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NusA is an 'E. coli' protein that controls transcription elongation, termination and antitermination. In this thesis, I show that the functions of NusA in transcription are facilitated by interactions with RNA polymerase, RNA and the [lambda] N protein. Use of a series of deletion constructs of NusA allowed me to identify specific regions of NusA involved in specific interactions and in various aspects of NusA function. Genetic evidence suggested that NusA may interact with the 'boxA ' portion of the N-utilization site ('nut' site= ' boxA, interbox,' and 'boxB'). By constructing multiple nucleotide substitutions in the 'nut' site, I showed that the identities of certain nucleotides at the 3' end of ' boxA' and in the 'interbox ' were important for NusA to associate with an N-'nut' site complex. NusA association with RNA in the presence of N is presumably facilitated by its S1 and KH homology regions, two types of RNA-binding domains in NusA. Elimination or mutation of the S1 homology region prevented the association of NusA with an N-' nut' site complex. Using affinity chromatography experiments, I found that RNA polymerase bound equally well to an amino-terminal RNA polymerase-binding region in amino acids 1-137 and a carboxy-terminal RNA polymerase-binding region in amino acids 232-495 of NusA. By contrast, the à subunit of RNA polymerase only bound to the carboxy-terminal RNA polymerase-binding region of NusA. N protein also bound to a carboxy-terminal region of NusA, and both N and à allowed NusA to associate with RNA in a gel mobility shift assay. When the carboxy-terminal region of NusA was deleted in NusA (1-348), the loss of N-binding and Ã-binding ability did not abolish NusA function in termination and antitermination assays. This minimal functional NusA protein retained the KH and S1 homology regions and the amino-terminal RNA polymerase-binding region. Unlike full length NusA (1-495), NusA (1-416) could bind RNA on its own. These observations suggest that the carboxy-terminal region of NusA inhibits RNA binding and that this inhibition can be relieved by interaction with the [lambda] N protein or the à subunit of RNA polymerase.
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Languages : en
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Author: Jingshu Guo
Publisher:
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Category :
Languages : en
Pages : 488
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Author: Asunción Martínez
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Category :
Languages : en
Pages : 466
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Author: Heeyoun Bunch
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Category :
Languages : en
Pages : 0
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Controlled gene expression in phage [lamda] is a model of transcription regulation which is mainly mediated by transcription factors to activate or repress E. coli RNA polymerase (RNAP). They switch appropriate genes on or off, determining the lysogenic or lytic pathway of progeny phages. Q is a [lamda] late gene regulator that exerts antitermination for products of the pR' promoter. Previous studies presented exciting aspects of [lamda]Q modification of RNAP which cause dramatic changes in the protein under certain conditions. RNAP engaged with [lamda]Q escapes a [sigma]-mediated promoter proximal pause and overcomes an intrinsic terminator downstream of the pR' promoter. In addition, [lamda]Q alters RNAP dynamics, aiding faster elongation, fewer pauses, and resistance to both the intrinsic and [rho]-dependent termination. Although [lamda]Q-mediated antitermination was characterized with previous works as described above, the mechanism by which [lamda]Q exerts antitermination is still unclear. In order to uncover this mechanism, this study attempted to answer the fundamental question of where [lamda]Q interacts with RNAP. First, the two largest subunits of RNAP were systematically dissected to map a [lamda]Q binding region on RNAP. The results suggested a clustered region of three fragments of [beta] and [beta]' for [lamda]Q binding, and as small as 82 amino acids ([beta]600-681) within this region were identified to bind to [lamda]Q. Second, a potential [lamda]Q binding region of RNAP proposed by a previous study was evaluated in this study. Although the region is located in the surface domain of [beta]600- 681, mutational analyses targeting the region demonstrated little [lamda]Q binding. Third, twelve RNAP mutants that reduce [lamda]Q-mediated antitermination were identified, and they suggested four spatial regions of RNAP as critical for [lamda]Q antitermination. Finally, one of the RNAP mutants, [beta]K1073G, presented altered function of NusA, a transcription elongation factor, in [lamda]Q-mediated antitermination at the intrinsic terminator, providing evidence of cooperation between NusA and [lamda]Q antitermination.
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Languages : en
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Author: Vladimir Uversky
Publisher: John Wiley & Sons
ISBN: 0470618310
Category : Science
Languages : en
Pages : 532
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This book provides up-to-date information on experimental and computational characterization of the structural and functional properties of viral proteins, which are widely involved in regulatory and signaling processes. With chapters by leading research groups, it features current information on the structural and functional roles of intrinsic disorders in viral proteomes. It systematically addresses the measles, HIV, influenza, potato virus, forest virus, bovine virus, hepatitis, and rotavirus as well as viral genomics. After analyzing the unique features of each class of viral proteins, future directions for research and disease management are presented.
Author: Alexandra Kimberly Smith
Publisher:
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Category :
Languages : en
Pages : 69
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Gene expression pathways exhibit many "twists and turns," with theoretically numerous ways in which the pathways can be regulated by both negative and positive feedback mechanisms. A key step in gene expression is RNA maturation (RNA processing), which in the bacterial cell can be accomplished through RNA binding and enzymatic cleavages. The well-characterized bacterial protein Ribonuclease III (RNase III), is a conserved, double-stranded(ds)-specific ribonuclease. In the gram-negative bacterium Escherichia coli, RNase III catalytic activity is subject to both positive and negative regulation. A recent study has indicated that an E. coli protein, YmdB, may negatively regulate RNase III catalytic activity. It has been proposed that YmdB inhibition of RNase III may be part of an adaptive, post-transcriptional physiological response to cellular stress. In E. coli, the model organism in this study, YmdB protein is encoded by the single ymdB gene, and has a predicted molecular mass of ~18.8 kDa. YmdB has been classified as a macrodomain protein, as it exhibits a characteristic fold that specifically provides an ADP-ribose (ADPR) binding site. While YmdB can bind ADPR with good affinity, there may be additional ligands for the binding site. Thus, YmdB protein may interact with other components in the cell, which in turn could modulate the interaction of YmdB with RNase III. In previous research conducted within the Nicholson laboratory at Temple University, affinity-purified Escherchia coli(Ec) YmdB and Aquifex aeolicus (Aa) YmdB were found to exhibit ribonucleolytic activity. This observation initiated the long-term goal of learning how YmdB regulates RNase III, and how the ribonucleolytic activity of YmdB may be involved in this process. The specific goal of this thesis project was to further characterize the ribonucleolytic activity of Ec-YmdB through site-specific mutational analysis. Mutations were introduced into a proposed adenine-binding pocket previously identified by crystallography and by molecular modeling. The adenine-binding pocket is a region within the macrodomain fold where ADP-ribose could bind. The mutations were examined for their effect on Ec-YmdB cleavage of a model RNA, R1.1. The results of this study will contribute to the development of a model describing how the ribonucleolytic activity of YmdB is regulated.
Author: Richard Calendar
Publisher: Oxford University Press
ISBN: 0195148509
Category : Science
Languages : en
Pages : 761
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This authoritative, timely, and comprehensively referenced compendium on the bacteriophages explores current views of how viruses infect bacteria. In combination with classical phage molecular genetics, new structural, genomic, and single-molecule technologies have rendered an explosion in our knowledge of phages. Bacteriophages, the most abundant and genetically diverse type of organism in the biosphere, were discovered at the beginning of the 20th century and enjoyed decades of used as anti-bacterial agents before being eclipsed by the antibiotic era. Since 1988, phages have come back into the spotlight as major factors in pathogenesis, bacterial evolution, and ecology. This book reveals their compelling elegence of function and their almost inconceivable diversity.Much of the founding work in molecular biology and structural biology was done on bacteriophages. These are widely used in molecular biology research and in biotechnology, as probes and markers, and in the popular method of assesing gene expression.
