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Degradation of Mrna Mk2 and Enhanced Transcription Termination Before the Galk Gene

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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 Xun Wang, Sang Chun Ji1, Heung Jin Jeon, Yonho Lee, and Heon M. Lim2 Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea Edited by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved May 11, 2015 (received for review December 29, 2014) The Escherichia coli gal operon has the structure Pgal-galE-galT- that there is an independent molecular mechanism other than galK-galM. During early log growth, a gradient in gene expression, transcription termination that operates to regulate galK expression named type 2 polarity, is established, as follows: galE > galT > galK > (1) and that the regulation of the amount of mK2 determines the galM. However, during late-log growth, type 1 polarity is established polarity type. in which galK is greater than galT,asfollows:galE > galK > galT > Small RNAs (sRNAs) in bacteria bind to their target mRNA galM. We found that type 2 polarity occurs as a result of the down- molecules and regulate their expression. Often, the binding of an regulation of galK, which is caused by two different molecular mech- sRNA to a target mRNA is mediated by Hfq, a homo-hexameric, anisms: Spot 42-mediated degradation of the galK-specific mRNA, bacterial Sm-like protein (3). In the case of an sRNA whose mK2, and Spot 42-mediated Rho-dependent transcription termination binding site on the target mRNA overlaps with the Shine- at the end of galT. Because the concentration of Spot 42 drops during Dalgarno (SD) sequence, the sRNA competes with the ribosome the transition period of the polarity type switch, these results dem- for binding. If sRNA binding dominates, it leads to the degrada- onstrate that type 1 polarity is the result of alleviation of Spot 42- tion of the target mRNA via the RNase E-mediated degradasome mediated galK down-regulation. Because the Spot 42-binding site (4). The binding of sRNA to the SD sequence of an mRNA overlaps with a putative Rho-binding site, a molecular mechanism is creates a ribosome-free region at the 5′-end of the mRNA be- proposed to explain how Spot 42, possibly with Hfq, enhances Rho- cause any translating ribosomes move away from the 5′ end, and mediated transcription termination at the end of galT. there is no de novo translation initiation (5). This ribosome-free region is suggested to be the substrate for the RNase E-mediated Spot 42 | galactose operon | Rho-termination | sRNA | degradasome (5). In some cases, if a Rho-dependent transcription target mRNA degradation termination signal resides in the ribosome-free region, binding of the sRNA leads to transcription termination by Rho (6). uring the exponential growth phase of Escherichia coli cells, An sRNA species known as Spot 42 was first recognized in Dtranscription termination at the end of each cistron of the 1973 from E. coli (7) and was later shown to be a 109-nucleotide- gal operon operates with less than 100% efficiency; a certain long primary transcript from the spf gene (8, 9). The cAMP-CRP proportion of the transcription initiated from the two promoters complex down-regulates the expression of the spf gene (10). The of the gal operon terminates at the end of each constituent gene, binding of Spot 42 to its target mRNAs down-regulates galK galE, galT, galK, and galM, of the operon (1). The termination expression (11). Spot 42 binding to a galK mRNA is mediated by efficiencies measured at the end of each cistron are 16%, 29%, Hfq (12). Spot 42 was shown to decrease GalK production, and it 65%, and 71%, respectively (1). Transcription termination at the was suggested that the binding of Spot 42 to the SD sequence in end of each cistron generates four mRNA species: mE1, mT1, galK inhibits galK translation (11). It was also demonstrated that mK1, and mM1 (Fig. 1). It has been demonstrated that RNase MICROBIOLOGY P-mediated internal cleavage of the premK2 transcript produces Significance the galK-specific mRNA, mK2, and it is assumed that the pro- moter-proximal part of the RNase P-cleaved premK2 becomes Most sRNAs of Escherichia coli function at the 5′ end of the mE2 (1) (Fig. 1). Thus, E. coli cells in the exponential growth target RNA. Binding of sRNA to the 5′ end of the target RNA phase would produce six mRNA species from the gal operon, as induces a ribosome-free zone that causes molecular events shown in Fig. 1. such as target RNA degradation and Rho-termination. Results Gene expression from each cistron has been measured using from this study show that Spot 42 enhances Rho-termination at real-time quantitative PCR (RT-qPCR) with a pair of primers the end of the galT gene, demonstrating for the first time that that binds to a specific region of each cistron, as shown in Fig. 1. sRNA could function at the 3′ end of the target RNA. The region Thus, the amount of transcript from each cistron (gene expres- where Spot 42 binds overlaps with two other functional cis-acting sion) is the sum of that of many mRNA species. For example, the sites: the ribosome-binding site for galK and the cytosine-rich, amount of transcript from galT is the sum of the amounts of guanine-poor region known as the Rho-binding site, suggesting a mE2, mT1, mK1, and mM1. The amount of transcript from galK unique molecular mechanism to enhance Rho-termination occur- is the sum of the amounts of mK1, mM1, and mK2 (Fig. 1). Thus, ring at a cistron junction in a multicistronic operon. the amount of transcript should be the greatest at the most promoter-proximal cistron and least at the most distal cistron, Author contributions: X.W. and H.M.L. designed research; X.W., S.C.J., H.J.J., and Y.L. with the pattern galE > galT > galK > galM; we have named this performed research; X.W. and H.M.L. analyzed data; and H.M.L. wrote the paper. the type 2 polarity (1). Thus, when one considers transcription The authors declare no conflict of interest. termination, only type 2 polarity should result. However, the This article is a PNAS Direct Submission. relative amounts of transcripts from WT cells at an optical 1Present address: Department of Clinical Pharmacology and Therapeutics, Seoul National density of 600 nm (A600) of 0.6, when actually measured, show a University College of Medicine and Hospital, Seoul 110-744, Korea. gradient in concentration that is galE > galK > galT > galM (1, 2). 2To whom correspondence should be addressed. Email: [email protected]. This type of polarity, where the amount of galK is always greater This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. than that of galT, was named type 1 polarity (1). We hypothesized 1073/pnas.1424683112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1424683112 PNAS | June 16, 2015 | vol. 112 | no. 24 | 7581–7586 Downloaded by guest on September 24, 2021 culture at each point. Total RNA isolated from each sampling was subjected to RT-qPCR, using four sets of PCR primers complementary to the end of each cistron of the gal operon. Results are shown in Fig. 2. The transcription dynamics for the overall production of the gal transcripts correspond very well with previously reported measurements (16, 17). It was demon- strated that the intracellular concentration of cAMP regulates the overall production of the gal transcripts by governing the rate of transcription initiation (17). We found that two types of po- larity, type 1 and type 2 (1), were established, depending on the growth period (Fig. 2). The polarity during early exponential Fig. 1. The gal operon and the mRNA species detected by Northern blot – > analysis (2) are presented as thick and thin lines, respectively. The numbers growth, at A600 values of 0.05 0.22, was type 2, in which galT indicate the positions of the stop codon of each cistron from the transcrip- galK. However, the polarity during late exponential growth, at tion initiation sites of the P1 promoter. The left-right arrows (↔) indicate the A600 values of 0.47–1.86, was type 1, in which galK > galT. During primer sets for RT-qPCR. Note that the primer sets are at the 3′ end of the the early exponential growth period (polarity type 2), the average respective genes, except in the case of galT. The stem and loop structure for expression ratio to galE was 0.4 for galT, 0.31 for galK, and 0.07 the transcription termination of mM1 is presented at the end of the operon. The for galM (Table S1). During late exponential growth (polarity three short lines under the mK1, mM1, and mK2 mRNA species indicate the type 1), however, the expression ratio to galE was 0.48 for galT, regions at which Spot 42 binds. The sequence of Spot 42 and its binding site at the galT–galK cistron junction are shown with short vertical lines in- 0.69 for galK, and 0.08 for galM (Table S1). These measurements dicating base-pairing. The stop codon of galT and the start codon of galK are showed that galK expression, which was 31% of galE in the early indicated in bold case. Hfq binds to the 3′ end of Spot 42 (12). The numbers exponential growth phase, increased to 69% in the late expo- indicate nucleotide positions.
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