Analysis of Corynebacterium glutamicum Promoters and Their Applications
Promoters are DNA sequences which function as regulatory signals of transcription initiation catalyzed by RNA polymerase. Since promoters substantially influence levels of gene expression, they have become powerful tools in metabolic engineering. Methods
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Analysis of Corynebacterium glutamicum Promoters and Their Applications Jan Nešvera, Jiří Holátko, and Miroslav Pátek
Abstract Promoters are DNA sequences which function as regulatory signals of transcription initiation catalyzed by RNA polymerase. Since promoters substantially influence levels of gene expression, they have become powerful tools in metabolic engineering. Methods for their localization used in Corynebacterium glutamicum and techniques for the analysis of their function are described in this review. C. glutamicum promoters can be classified according to the respective s factors which direct RNA polymerase to these structures. C. glutamicum promoters are recognized by holo-RNA polymerase formed by subunits a2bb¢w + s. C. glutamicum codes for seven different sigma factors: the principal sigma factor sA and alternative sigma factors sB, sC, sD, sE, sH and sM, which recognize various classes of promoters. The promoters of housekeeping genes recognized by sA, which are active during the exponential growth, form the largest described group. These promoters and their mutant derivatives are the most frequently used elements in modulation of gene expression in C. glutamicum. Promoters recognized by alternative sigma factors and their consensus sequences are gradually emerging. Keywords Corynebacterium glutamicum • Gene expression • Promoter • Promoter consensus sequence • Sigma factor
Abbreviations RACE RNAP ROMA
rapid amplification of cDNA-ends RNA polymerase run-off transcription-microarray analysis
J. Nešvera • J. Holátko • M. Pátek (*) Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic e-mail: [email protected] X. Wang et al. (eds.), Reprogramming Microbial Metabolic Pathways, Subcellular Biochemistry 64, DOI 10.1007/978-94-007-5055-5_10, © Springer Science+Business Media Dordrecht 2012
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TSP WT
10.1
J. Nešvera et al.
transcriptional start point wild-type
Introduction
Transcription initiation is the major regulatory step in gene expression in bacteria. Promoters are the specific sequences within the noncoding regulatory regions of genes which are recognized by RNA polymerase (RNAP) and determine thus the location of the transcriptional start points. The sequence of a promoter can be crucial for the frequency of transcription initiation and consequently for the level of gene expression. Since a promoter is formed by a short DNA sequence, whose regulatory functions can be described in detail, it is an attractive and efficient element in the armory of tools for metabolic engineering. The choice of constitutive or inducible promoters still widens the possibilities for a strategy of purposeful controlled gene expression. Knowledge of promoters and their regulation in a particular organism is necessary for rational modulation of gene expression and for the application of these powerful tools in metabolic engineering. Promoters of C. glutamicum, a widely used industrial producer of amino acids, play an important role in mole
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