Establishment of an mRNA Gradient Depends on the Promoter: An Investigation of Polarity in Gene Expression

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

doi:10.1016/j.jmb.2008.02.067

Cited by 33 publications

(50 citation statements)

References 34 publications

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“…Next, we performed Northern blot assays on equal amounts of total RNA from pBRlac/WT and pSpot42/WT cells, using the T probe, which is complementary to the first half of the galT gene (2). With this probe, we could detect all of the gal mRNA species except mK2 (i.e., mE1, mE2, mT1, mK1, and mM1).…”

Section: Spot 42 Enhances Rho-mediated Transcription Termination At Thementioning

confidence: 99%

“…Thus, when one considers transcription termination, only type 2 polarity should result. However, the relative amounts of transcripts from WT cells at an optical density of 600 nm (A 600 ) of 0.6, when actually measured, show a gradient in concentration that is galE > galK > galT > galM (1,2). This type of polarity, where the amount of galK is always greater than that of galT, was named type 1 polarity (1).…”

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confidence: 99%

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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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Section: Spot 42 Enhances Rho-mediated Transcription Termination At Thementioning

confidence: 99%

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confidence: 99%

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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“…+ has been described (14,15). Strains were grown to an OD600 of 0.6 in LB containing 0.5% galactose at 37 o C.…”

Section: P2mentioning

confidence: 99%

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

Wang²,

Jeon³

et al. 2010

BMB Reports

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Fig. 1. A schematic presentation of the galactose operon. The -10 regions of the P1 and P2 promoters are shown in boxes. The transcription initiation sites are indicated by arrows and underlined. CRP points to its DNA binding site. OE and OI designate the operators where GalR binds. HU protein binds to hbs. The transcription initiation site of the P1 promoter is assigned as +1, Nucleotide positions of the operon, thus, have been assigned relative to the P1 initiation site.

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“…A Northern blot analysis showed that there are 6 different species of mRNA specific to the galactose operon in wild-type E. coli cells grown exponentially in the presence of galactose (2). Five of the 6 mRNA species, mE1, mE2, mT1, mK1, and mM1, have their 5= ends at the transcription initiation region, and their 3= ends at 5 different locations within the operon, four of which (all but mE2) are at the ends of the galE, galT, galK, and galM cistrons, respectively (Fig.…”

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confidence: 99%

“…The existence of these mRNA species automatically establishes a gradient of gene expression, higher in the promoter-proximal region and lower in the promoter-distal region, which has been referred to as "natural polarity" (3). Natural polarity is intrinsically different from what has been known as polarity that is caused by a mutation (4), because it can be observed in cells harboring the wild-type operon (2,(5)(6)(7)(8)(9). The term "polarity" refers to the phenomenon in which a mutation in one gene of an operon decreases the expression of the subsequent genes of the operon.…”

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Expression of Each Cistron in the gal Operon Can Be Regulated by Transcription Termination and Generation of a galK -Specific mRNA, mK2

Wang

Yun

et al. 2014

J Bacteriol

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bThe gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378:318 -327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3= ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5= ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galEgalT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5= ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3= ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5= portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity. P olycistronic operons in bacteria show a differential expression of the constituent cistrons (1). A Northern blot analysis showed that there are 6 different species of mRNA specific to the galactose operon in wild-type E. coli cells grown exponentially in the presence of galactose (2). Five of the 6 mRNA species, mE1, mE2, mT1, mK1, and mM1, have their 5= ends at the transcription initiation region, and their 3= ends at 5 different locations within the operon, four of which (all but mE2) are at the ends of the galE, galT, galK, and galM cistrons, respectively (Fig. 1A). There is one distinct mRNA species, designated mK2, that has 5= ends not at the promoter region but at the middle of galT. The existence of these mRNA species automatically establishes a gradient of gene expression, higher in the promoter-proximal region and lower in the promoter-distal region, which has been referred to as "natural polarity" (3). Natural polarity is intrinsically different from what has been known as polarity that is caused by a mutation (4), because it can be observed in cells harboring the wild-type operon (2, 5-9). The term "polarity" refers to the phenomenon in which a mutation in one gene of an operon decreases the expression of the subsequent genes of the operon. The cause for polarity is well established. The cessation of translation by a nonsense mutation uncouples transcription from translation, allowing the transcription termination factor, Rho, to bind to the nascent RNA and terminate transcription at the next available termination signal. This Rho-mediated transcription termination leaves the rest of the operon untranscribed, creating polarity ...

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Establishment of an mRNA Gradient Depends on the Promoter: An Investigation of Polarity in Gene Expression

Cited by 33 publications

References 34 publications

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

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

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

Expression of Each Cistron in the gal Operon Can Be Regulated by Transcription Termination and Generation of a galK -Specific mRNA, mK2

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