Characterizing a Promoter Specificity Determinant of Bacteriophage T7 and T3 RNA Polymerases PDF Download
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Author: Curtis A. Raskin
Publisher:
ISBN:
Category :
Languages : en
Pages : 156
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Author: Curtis A. Raskin
Publisher:
ISBN:
Category :
Languages : en
Pages : 156
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Author: John F. Klement
Publisher:
ISBN:
Category : Bacteriophages
Languages : en
Pages : 410
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Author: Minqing Rong
Publisher:
ISBN:
Category :
Languages : en
Pages : 336
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Author: Claire Ellen Morris
Publisher:
ISBN:
Category : Bacteriophages
Languages : en
Pages : 410
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Author:
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ISBN:
Category :
Languages : en
Pages :
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Book Description
This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.
Author: Biao He
Publisher:
ISBN:
Category :
Languages : en
Pages : 438
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Author: Ramesh Padmanabhan
Publisher:
ISBN:
Category : Bacteriophages
Languages : en
Pages :
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The bacteriophage T7 RNA polymerase (T7 RNAP) is the prototype of the family of single subunit RNA polymerases which includes the T3, SP6 and mitochondrial RNA polymerases. It is also the most well characterized enzyme of this family of polymerases. T7 RNAP is the primary choice in studying the mechanistic aspects of transcription and promoter evolution owing to its high specificity for its promoter, requirement of no additional cofactors, and high fidelity of initiation from a specific site in its promoter. Although the two groups of single subunit polymerases, bacteriophage and mitochondrial, display a remarkable structural conservation, they recognize quite dissimilar promoters. Functional domains involved in promoter recognition and transcription initiation have been well characterized by thorough mutational studies by systematic deletions within these domains. T7 RNAP recognizes a 23 nucleotide (nt) promoter which can be divided into several functional domains. T7 RNAP can recognize a range of promoter sequences which are closely related to this consensus sequence. The promoter-specific recognition is achieved by the 13 nucleotide promoter recognition region which extends from -5 to -17 and contains sites important for polymerase recognition and positioning. Here, we have developed an in vitro transcription system to study the ability of T7 RNAP to use truncated promoters similar to mitochondrial promoters. In this system, we used oligonucleotides which are capable of forming intra- and inter-molecular base pairing to produce a recessed 3’end on an extended 5’ template. We then systematically deleted nucleotides from the 5’ end of the 20-nucleotide double stranded region on these oligonucleotides containing the T7 RNAP promoter to determine (1) If they would label at the 3’ end of the oligonucleotide, (2) Initiate template-dependent de novo transcription, (3) Initiate template-independent and promoter-dependent de novo transcription or, (4) Fail to incorporate label when supplied with T7 RNAP and a single radiolabeled ribonucleotide triphosphate, which can base pair with the first unpaired base in the 5’ extension of the template. Under this condition, when a complete or almost complete (20 to 16 nt) double stranded T7 RNAP promoter is present, small RNAs are produced through template-independent and promoter-dependent stuttering corresponding to abortive initiation, which is lost when supplied with a completely scrambled promoter sequence. When partial double stranded promoter sequences (10-12 nt) are provided, template dependent de novo initiation of RNA occurs but at a site different from the canonical +1 initiation site (-GGG). In situations where no promoter is present but the potential exists to form a transient duplex region with a recessed 3’ end, the T7 RNAP adds a templated nucleotide to the 3’ end (primer labeling). Understanding the mechanisms underlying these observations helps us to understand the roles played by promoter elements. The evolution of promoter sequences of the single subunit RNAPs and to use this as a technique to synthesize defined RNAs without 5’- sequence constraints are discussed.
Author: Gnana Sakuntala Warshamana
Publisher:
ISBN:
Category : RNA polymerases
Languages : en
Pages : 306
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Author: Joseph M. Fernandez
Publisher: Elsevier
ISBN: 0080532357
Category : Science
Languages : en
Pages : 493
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Book Description
Gene Expression Systems: Using Nature for the Art of Expression offers detailed information on a wide variety of gene expression systems from an array of organisms. It describes several different types of expression systems including transient, stable, viral, and transgenic systems. Each chapter is written by a leader in the field. The book includes timelines and examples for each expression system, and provides an overview of the future of recombinant protein expression. - Provides detailed information on expression systems - Covers a variety of promoters and host organisms enabling researchers to tailor protocols to their specific needs - Includes timelines and examples - Compares pros and cons of each method
Author: Richard Calendar
Publisher: Oxford University Press
ISBN: 0195148509
Category : Science
Languages : en
Pages : 761
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Book Description
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.
