Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate 'green waves' in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biotechnologie metody   MeSH
• Corynebacterium glutamicum genetika   metabolismus   MeSH
• genetická transkripce MeSH
• konsenzuální sekvence MeSH
• metabolické sítě a dráhy MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH

Novel shuttle promoter-probe vectors replicating in Escherichia coli, Corynebacterium glutamicum, and Rhodococcus erythropolis were constructed on the basis of the C. glutamicum plasmid pCG1. The vectors carry reporter genes coding for fluorescent proteins, which allow the measurement of promoter activities in vivo. The promoter-probe vector pPRE11 contains the rsgfp reporter gene, coding for a variant of green fluorescent protein (GFP) with a red-shifted excitation maximum. To ensure efficient expression of the gfp gene in R. erythropolis from the tested promoters, the promoterless gene gfpuv, with 5' end fusion with the initial six codons of the aph gene and upstream insertion of the aph Shine-Dalgarno sequence, was used as a reporter gene in the promoter-probe vector pEPR1. Insertion of the rfp reference gene, coding for a variant of the red fluorescent protein DsRed.T4 and cloned under the strong constitutive C. glutamicum promoter P-45, into the vector pEPR1 resulted in a new-generation promoter-probe vector (pRAG5). All vectors were tested using a set of mutant P-dapA promoters displaying various transcriptional activities. The vector pRAG5 is suitable for normalized measurements of promoter activities during the growth of bacterial batch cultures because estimation of the GFP-to-red fluorescent protein fluorescence ratio in strains carrying the plasmid pRAG5 with the tested promoters upstream of gfpuv avoids the influence of plasmid copy number variations on the promoter activity assay.

• MeSH
• Corynebacterium glutamicum genetika   MeSH
• genetické vektory * MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• Rhodococcus genetika   MeSH
• zelené fluorescenční proteiny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH