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Strength and Regulation of Seven rRNA Promoters in Escherichia coli.

Maeda M, Shimada T, Ishihama A - PLoS ONE (2015)

Bottom Line: Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector.These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations.Possible difference in the functional role of each rRNA operon is also discussed.

View Article: PubMed Central - PubMed

Affiliation: Meiji University, Faculty of Agriculture Chemistry, Kawasaki, Kanagawa 214-8571, Japan.

ABSTRACT
The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed.

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Growth rate-dependent variation of the promoter activity of seven rrn operons.The promoter activity of seven rrn operons of E. coli was separately determined as described in Fig 5. [A] The ratio between the cell growth rate in four different culture media and the highest level of promoter activity in the middle of exponential growth phase is plotted for each of seven rrn promoters. [B] The highest level of promoter activity in four different cultures is shown for each of seven rrn operon promoters. The activity of rrnE promoter was the highest under all the culture conditions employed.
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pone.0144697.g008: Growth rate-dependent variation of the promoter activity of seven rrn operons.The promoter activity of seven rrn operons of E. coli was separately determined as described in Fig 5. [A] The ratio between the cell growth rate in four different culture media and the highest level of promoter activity in the middle of exponential growth phase is plotted for each of seven rrn promoters. [B] The highest level of promoter activity in four different cultures is shown for each of seven rrn operon promoters. The activity of rrnE promoter was the highest under all the culture conditions employed.

Mentions: For construction of the promoter assay vectors of rrn operons, a total of approximately 500 bp-long sequence upstream from 5’ terminus of 16S rRNA gene, as indicated above each DNA lane, were PCR-amplified using specific set of primers (for primer sequences see S1 Table) and inserted into pGRS vector, a modified form of pGRP [28], containing an SD sequence at the junction of GRF-coding sequence. Open box and closed boxes on each probe represent the relative location of UP element and predicted Fis-binding sites, respectively (see Fig 8). Triangles downstream of the UP element indicate two promoters, upstream P1 and downstream P2. The number of Fis sites on the rrnE promoter was suggested to be more than those hitherto identified (see Fig 3). The whole length used for the construction of pGRS vector is described in parenthesis at right-side end of each lane.


Strength and Regulation of Seven rRNA Promoters in Escherichia coli.

Maeda M, Shimada T, Ishihama A - PLoS ONE (2015)

Growth rate-dependent variation of the promoter activity of seven rrn operons.The promoter activity of seven rrn operons of E. coli was separately determined as described in Fig 5. [A] The ratio between the cell growth rate in four different culture media and the highest level of promoter activity in the middle of exponential growth phase is plotted for each of seven rrn promoters. [B] The highest level of promoter activity in four different cultures is shown for each of seven rrn operon promoters. The activity of rrnE promoter was the highest under all the culture conditions employed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696680&req=5

pone.0144697.g008: Growth rate-dependent variation of the promoter activity of seven rrn operons.The promoter activity of seven rrn operons of E. coli was separately determined as described in Fig 5. [A] The ratio between the cell growth rate in four different culture media and the highest level of promoter activity in the middle of exponential growth phase is plotted for each of seven rrn promoters. [B] The highest level of promoter activity in four different cultures is shown for each of seven rrn operon promoters. The activity of rrnE promoter was the highest under all the culture conditions employed.
Mentions: For construction of the promoter assay vectors of rrn operons, a total of approximately 500 bp-long sequence upstream from 5’ terminus of 16S rRNA gene, as indicated above each DNA lane, were PCR-amplified using specific set of primers (for primer sequences see S1 Table) and inserted into pGRS vector, a modified form of pGRP [28], containing an SD sequence at the junction of GRF-coding sequence. Open box and closed boxes on each probe represent the relative location of UP element and predicted Fis-binding sites, respectively (see Fig 8). Triangles downstream of the UP element indicate two promoters, upstream P1 and downstream P2. The number of Fis sites on the rrnE promoter was suggested to be more than those hitherto identified (see Fig 3). The whole length used for the construction of pGRS vector is described in parenthesis at right-side end of each lane.

Bottom Line: Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector.These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations.Possible difference in the functional role of each rRNA operon is also discussed.

View Article: PubMed Central - PubMed

Affiliation: Meiji University, Faculty of Agriculture Chemistry, Kawasaki, Kanagawa 214-8571, Japan.

ABSTRACT
The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed.

Show MeSH
Related in: MedlinePlus