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

Abstract

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 ...

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

“…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.…”

“…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).…”

“…1). It has been demonstrated that RNase P-mediated internal cleavage of the premK2 transcript produces the galK-specific mRNA, mK2, and it is assumed that the promoter-proximal part of the RNase P-cleaved premK2 becomes mE2 (1) (Fig. 1).…”

“…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).…”

“…This type of polarity, where the amount of galK is always greater than that of galT, was named type 1 polarity (1). We hypothesized that there is an independent molecular mechanism other than transcription termination that operates to regulate galK expression (1) and that the regulation of the amount of mK2 determines the polarity type.…”