Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

Ji, Sang Chun; Wang, Xun; Jeon, Heung Jin; Yun, Sang Hoon; Lee, Hee Jung; Lim, Heon M.

doi:10.5483/bmbrep.2010.43.7.474

Cited by 3 publications

(4 citation statements)

References 15 publications

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“…4A) [20]. The relative number of P1 -initiated transcripts was calculated by subtracting the number of P2 transcripts from the total number of transcripts.…”

Section: Resultsmentioning

confidence: 99%

“…To ligate the 3′-hydroxyl end of 5S rRNA to the 5′ phosphate end of gal mRNA, a 25-µl reaction containing 10 µl total RNA, 2.5 µl 10× reaction buffer (Ambion), 10 units T4 RNA ligase (Ambion), and 20 units RNasin (Promega) was incubated at 37°C for 4 h. This RNA was purified with a G-50 column (Amersham Biosciences). Using this RNA preparation, real-time reverse transcription-polymerase chain reaction (RT-PCR) and primer extension were performed as previously described [20]. The 5′ RACE assay was used to distinguish mRNAs transcribed from P1 (70-bp) from those transcribed from P2 (75-bp), which differs by five bases at the 5′ end.…”

Section: Methodsmentioning

confidence: 99%

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In Vivo Transcription Dynamics of the Galactose Operon: A Study on the Promoter Transition from P1 to P2 at Onset of Stationary Phase

Wang

Yun

et al. 2011

PLoS ONE

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Quantitative analyses of the 5′ end of gal transcripts indicate that transcription from the galactose operon P1 promoter is higher during cell division. When cells are no longer dividing, however, transcription is initiated more often from the P2 promoter. Escherichia coli cells divide six times before the onset of the stationary phase when grown in LB containing 0.5% galactose at 37°C. Transcription from the two promoters increases, although at different rates, during early exponential phase (until the third cell division, OD600 0.4), and then reaches a plateau. The steady-state transcription from P1 continues in late exponential phase (the next three cell divisions, OD600 3.0), after which transcription from this promoter decreases. However, steady-state transcription from P2 continues 1 h longer into the stationary phase, before decreasing. This longer steady-state P2 transcription constitutes the promoter transition from P1 to P2 at the onset of the stationary phase. The intracellular cAMP concentration dictates P1 transcription dynamics; therefore, promoter transition may result from a lack of cAMP-CRP complex binding to the gal operon. The decay rate of gal-specific transcripts is constant through the six consecutive cell divisions that comprise the exponential growth phase, increases at the onset of the stationary phase, and is too low to be measured during the stationary phase. These data suggest that a regulatory mechanism coordinates the synthesis and decay of gal mRNAs to maintain the observed gal transcription. Our analysis indicates that the increase in P1 transcription is the result of cAMP-CRP binding to increasing numbers of galactose operons in the cell population.

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“…4A) [20]. The relative number of P1 -initiated transcripts was calculated by subtracting the number of P2 transcripts from the total number of transcripts.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In Vivo Transcription Dynamics of the Galactose Operon: A Study on the Promoter Transition from P1 to P2 at Onset of Stationary Phase

Wang

Yun

et al. 2011

PLoS ONE

Self Cite

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“…Total RNA from the culture was prepared, as described in [20]. Total RNA (2 μg) was reverse transcribed in a 20-μl reaction containing 4 U Omniscript Reverse Transcriptase (QIAGEN), 0.5 mM each dNTP, 10 µM random nonamer (Takara Bio, Shiga, Japan), and 10 U RNasin.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA (2 μg) was reverse transcribed in a 20-μl reaction containing 4 U Omniscript Reverse Transcriptase (QIAGEN), 0.5 mM each dNTP, 10 µM random nonamer (Takara Bio, Shiga, Japan), and 10 U RNasin. PCR amplification of a specific cDNA was performed in a 20-μl reaction using 1 μl cDNA [20]. Real-time qPCR was performed in a 10-μl reaction containing 5 μl iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA), 3.6 μl nuclease-free water, 0.2 μl each primer (10 µM), and 1 μl cDNA template under the following conditions: an initial denaturation step at 95°C for 3 min; 40 cycles of 10 s of denaturation at 94°C, 20 s of hybridization at 56°C, and 15 s of elongation at 72°C (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

The CnuK9E H-NS Complex Antagonizes DNA Binding of DicA and Leads to Temperature-Dependent Filamentous Growth in E. coli

Yun

Jeon

et al. 2012

PLoS ONE

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Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25°C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37°C, presumably due to the lower operator binding of DicA at 37°C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25°C.

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Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

Wang

Jeon

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The Escherichia coli gal operon has the structure Pgal-galE-galTgalK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the downregulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rhomediated transcription termination at the end of galT.D uring the exponential growth phase of Escherichia coli cells, transcription termination at the end of each cistron of the gal operon operates with less than 100% efficiency; a certain proportion of the transcription initiated from the two promoters of the gal operon terminates at the end of each constituent gene, galE, galT, galK, and galM, of the operon (1). The termination efficiencies measured at the end of each cistron are 16%, 29%, 65%, and 71%, respectively (1). Transcription termination at the end of each cistron generates four mRNA species: mE1, mT1, mK1, and mM1 (Fig.

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Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

Cited by 3 publications

References 15 publications

In Vivo Transcription Dynamics of the Galactose Operon: A Study on the Promoter Transition from P1 to P2 at Onset of Stationary Phase

In Vivo Transcription Dynamics of the Galactose Operon: A Study on the Promoter Transition from P1 to P2 at Onset of Stationary Phase

The CnuK9E H-NS Complex Antagonizes DNA Binding of DicA and Leads to Temperature-Dependent Filamentous Growth in E. coli

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

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