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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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 promoter-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 30, 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 demonstrated 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 polarity, 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. nential growth period, whereas the expression of galT and galM increased only slightly. These results suggest there is a substantial increase in galK expression alone that stimulates the switch in the level of galK transcript decreases if Spot 42 is overexpressed polarity from type 2 to type 1. (13), suggesting that Spot 42 is also engaged in galK down-regulation at the transcriptional level. However, the logic that Spot 42-Mediated galK Down-Regulation Is Alleviated in the Late prompted this investigation was that the Spot 42 binding sites Exponential Growth Period. We also measured the amount of present in the three target mRNAs (Fig. 1) should lead to dif- Spot 42 at different times of growth. Total RNA was prepared ferent effects. The Spot 42 binding site on mK2 resides at its 5′ from each sampling presented earlier and subjected to RT-qPCR end, whereas those on mK1 and mM1 are in the middle of the to measure the amount of Spot 42. Results showed that the – polycistronic mRNA known as the galT galK junction. At this amount of Spot 42 reached a maximum as early as at an A600 junction, the stop codon of galT is separated by three nucleotides value of 0.05 and stayed at this maximum for the next two from the start codon of galK, and the translation of galT is doubling times, with A600 values of 0.22. At the third doubling coupled to that of galK (14). Thus, the junction should be oc- time, at an A600 value of 0.47, there was a sudden drop in the cupied by translating ribosomes. In addition, Rho-termination amount of Spot 42 to 25% of the initial concentration (Fig. 3). occurs at the galT–galK junction to generate a gal-specific Taken together, these data demonstrate that during early expo- mRNA, mT1 [Fig. 1 (1)]. These considerations led us to hy- nential growth, when the concentration of Spot 42 is highest, the pothesize that Spot 42 would use different molecular mecha- polarity is type 2. However, during late exponential growth, when nisms to inhibit galK expression (i.e., other than the inhibition of Spot 42 concentration is maintained at 25% of its maximum, galK translation) (11). type 1 polarity results. These observations suggest that Spot 42 Often, constituent cistrons in a bacterial polycistronic operon functions in vivo to down-regulate galK expression during early exhibit differential expression (15). In this study, we investigated exponential growth. Indeed, Spot 42 has been shown to how Spot 42 down-regulates galK expression, Northern blot assays of gal mRNAs from cells overexpressing Spot 42 suggested that Spot 42 leads to the degradation of mK2 and enhances transcription termination at the end of the galT gene (i.e., before the galK gene). Measurements of gal mRNA levels showed that both mechanisms, mK2 degradation and transcription termination, result in the down-regulation of galK expression only in the early exponential growth period (A600 of 0.2) when the concentration of Spot 42 is high. These results explain why the amount of galK transcript is higher than that of galT during the later exponential growth period (A600 of 0.6), despite the fact that galT is more proximal to the promoters of the operon, demonstrating that Spot 42 determines the type of polarity by regulating the concentration of mK2. Results Expression of galK Relative to galE Increases in the Late Exponential Growth Period. We measured the mRNA concentration gradient Fig. 2. The expression levels of galE, galT, galK, and galM at different times [polarity (1)] of gal-specific transcripts at different times of of growth in WT cells are presented in the bar graph. Cells were grown in LB growth. Unless noted, we have performed experiments on total medium supplemented with 0.5% galactose. The growth of the culture from × 8 which the samples were drawn is represented by the thin line. Expression RNA isolated from 2 10 cells grown in LB medium supple- levels were measured from a total RNA preparation at the indicated times, mented with 0.5% galactose throughout this study. By taking half using RT-qPCR, with the four sets of primers represented by the left-right the volume of the previous sampling at every doubling time, we arrows in Fig. 1. The measurements were repeated three times, and SDs 8 were able to harvest the same number of cells (2 × 10 ) from the are presented.

7582 | www.pnas.org/cgi/doi/10.1073/pnas.1424683112 Wang et al. Downloaded by guest on September 30, 2021 1764th and 1777th nucleotides from the P1 promoter initiation site (1). Thus, the Spot 42 binding site on mK2 is now located near the 5′ end of mK2 and overlaps with the SD sequence of galK in mK2. Because the binding of many sRNA species to the SD of the target mRNA leads to degradation of the target mRNA (4), we considered it likely that Spot 42 binding to mK2 could lead to the degradation of mK2. This model was tested. We used a plasmid, pSpot42, that could overexpress Spot 42 under the control of the lac promoter (13). The expression of Spot 42 from this plasmid was so leaky that a significant amount of Spot 42 (43% of the amount at full induction) was expressed even without induction (Fig. S1). We measured the expression of mK2, mK1, and mM1 by Northern blot assay in WT cells harboring pBRlac, a control plasmid for pSpot42 (pBRlac/WT), and in WT cells harboring the pSpot42 (pSpot42/WT), using the K probe. This K probe, which is complementary to the first half of Fig. 3. The expression of Spot 42 RNA at different times of growth in WT the galK gene, detects mK2, mK1, and mM1 (2). Results showed cells is represented by the dark line. The growth of the culture from which the samples were drawn is represented by the gray line. Expression levels that the level of mK2 almost disappears in pSpot42-harboring were measured from a total RNA preparation at the indicated times, using cells that overexpress Spot 42, whereas mK1 is slightly reduced RT-qPCR. The measurements were repeated three times. and the change in mM1 is marginal (Fig. 5), suggesting that Spot 42 does indeed specifically degrade mK2. These results support our notion that the effect of Spot 42 binding at mK1 or mM1 is down-regulate the amount of galK transcript (13). Therefore, the likely different from the effect at mK2, which is degradation. molecular mechanism that operates exclusively to increase galK These results also suggest that the effect of Spot 42 on mK1 or expression might be the alleviation of Spot 42-mediated down- mM1 seemed minor compared with the effect on mK2. Thus, the regulation with the decrease in Spot 42 concentration. molecular mechanism underlying the down-regulation of galK expression by Spot 42 is degradation of mK2 on Spot 42 binding, The Degree of Increase of mK2 Is Greater Than Those of mK1 and and possibly another mechanism (see following). mM1 When Spot 42 Is Absent. The cAMP-CRP complex is a transcriptional factor that negatively regulates the expression of Spot 42 Has a Pivotal Role in the Generation of mT1 3′ Ends. The Spot 42 (10). A comparison of the concentration change of effect of degradation of mK2 is understandable because the Spot cAMP (17) versus Spot 42 (Fig. 3) at different times of growth 42 binding site is now at the 5′ end of the target mRNA, and the reveals an inverse relationship: When the intracellular concen- binding of Spot 42 would create a ribosome-free zone. Fur- tration of cAMP starts to increase, that of Spot 42 starts to de- thermore, this kind of effect of sRNA on the target mRNA has crease. Thus, the polarity of an adenylyl cyclase mutant strain been demonstrated in several cases (18–21). However, the should be type 2 because Spot 42 concentration in the adenylyl binding of Spot 42 in mK1 and mM1 was very hard to compre- cyclase mutant strain would be maintained at a higher level than hend because the Spot 42 binding site is in the middle of a Δ in WT. Indeed, when we measured polarity in the MG1655 cya polycistronic mRNA, and presumably under a continuous flow of strain at an A600 value of 0.6, it was type 2 (Fig. 4A). Note that in translating ribosomes. In mK1 or mM1, translation initiation of WT cells, the polarity at an A600 value of 0.6 was type 1 (Fig. 2). galK is known to be a continuous process of translation termi- Δ Thus, we reasoned that if Spot 42 is removed from the cya nation at the end of galT before translational coupling (14). strain, then the type 2 polarity would switch to type 1. We de- Thus, we envisioned that Spot 42 might engage in binding to its leted the spf gene, from which Spot 42 is transcribed, from the

binding site that has just been synthesized, instead of binding to MICROBIOLOGY chromosome of the Δcya strain to construct MG1655 Δcya Δspf. Δ Δ the full-length mK1 or mM1 mRNA. Therefore, Spot 42 binding As expected, the polarity in MG1655 cya spf at an A600 value might be engaged in transcription termination instead of trans- of 0.6 was switched to type 1 (Fig. 4A). lation inhibition. To test this model, we attempted to visualize The amount represented as galK in the polarity measurement the 3′ ends of mT1 to assay transcription termination at the galT-K is the sum of the quantities of each of the three transcripts mK1, mK2, and mM1. To investigate how Spot 42-mediated galK down-regulation is lifted, we analyzed the expression of the three gal mRNAs from the MG1655 Δcya and MG1655 Δcya Δspf strains by Northern blot analysis. Total RNA was prepared from the cultures at an A600 value of 0.6, and the same amounts were subjected to Northern blot analysis. The Northern blot was probed with the K primer that is complementary to the first half of the galK gene. The results showed that the quantities of each of the three mRNAs were increased in the double mutant, MG1655 Δcya Δspf (Fig. 4B), demonstrating that lifting Spot 42-mediated galK down-regulation results in an increase in the amount of each of the three mRNAs. However, there was a differential increment: mK2 increased more than fourfold, whereas mK1 and mM1 increased approximately twofold in Δcya Fig. 4. (A) The expression levels of galE, galT, galK, and galM from the Δspf cells, further suggesting that mK2 can be generated in dif- MG1655 Δcya and MG1655 Δcya Δspf strains are presented in the bar graph. ferent amounts than mK1 or mM1. Total RNA prepared from the culture at A600 of 0.6 was subjected to tripli- cate RT-qPCR measurements. (B) Northern blot analysis with the K probe in Spot 42 Causes mK2 Degradation. We showed in our previous re- MG1655 Δcya and MG1655 Δcya Δspf strains. The RNAs were visualized port that the 5′ end of mK2 is generated by RNase P-mediated using the K probe labeled with the DIG system (Roche). Note that the K endoribonucleolytic cleavage of a gal transcript between the probe detects mK2, mK1, and mM1 only.

Wang et al. PNAS | June 16, 2015 | vol. 112 | no. 24 | 7583 Downloaded by guest on September 30, 2021 2169, they were reduced to 10% of WT (Fig. 6B). These results demonstrated that Hfq plays an important role in Rho-mediated transcription termination at the galT–galK cistron junction. Be- cause the 3′ ends are down-regulated in the hfq mutant strain, these results suggested that Hfq may have a role in transcription termination, possibly in promoting Rho-termination.

Spot 42 Enhances Rho-Mediated Transcription Termination at the galT–galK Junction. Thus far, the effect of Spot 42 on galK expression was seen to be negative, with down-regulation of GalK production (11), decreased levels of the galK transcript (13), and degradation of mK2 (Fig. 5). We hypothesized that the “other mechanism” of galK down-regulation by Spot 42 suggested from Fig. 5 could involve enhancing transcription termination at the galT–galK junction. In addition to the clear demonstration of mK2 being degraded, we observed that mK1 and mM1 were slightly down-regulated by Spot 42 (Fig. 5). To demonstrate further that Spot 42 could enhance transcription termination at Fig. 5. Northern blot analysis with the K probe in WT cells harboring the – 32 the galT galK junction, we investigated the effect of Spot 42 on plasmid pBRlac or pSpot42. The RNAs were visualized using the P -labeled K mT1 3′ ends. If the above hypothesis is accurate, the amount of probe. The band right above mK2 is unknown but appears to be related to mT1 3′ ends would be expected to increase if Spot 42 is over- 23S rRNA, judging from the fact that the space between mK1 and the unknown ′ ′ band is where 23S rRNA resolves. We have noticed that this nonspecific band expressed. We performed 3 RACE and visualized the 3 ends appears when fragmentation of RNA in agarose gel by UV radiation is overdone. of mT1 in WT cells harboring the pSpot42 plasmid and over- Note that the K probe detects mK2, mK1, and mM1 only. expressing Spot42 (pSpot42/WT). The results showed that only the bands that represent the 3′ ends of mT1 (from +2124 to +2169) increased twofold in pSpot42/WT cells compared with cistron junction, using a 3′ random amplification of cDNA ends the 3′ ends of mT1 in the control strain, pBRlac/WT (Fig. 7A), (RACE) assay. Briefly, total RNA was subjected to RNA ligation thus demonstrating that Spot 42 promotes transcription termi- to a 20-mer synthetic RNA. Subsequently, cDNA synthesis was nation at the galT–galK junction. performed with a DNA primer complementary to the synthetic Next, we performed Northern blot assays on equal amounts of RNA (primer A). Because of the orientation of the 3′ end of the total RNA from pBRlac/WT and pSpot42/WT cells, using the T synthetic RNA, cDNA synthesis only occurs on those mRNAs probe, which is complementary to the first half of the galT gene whose 3′ ends are ligated to the synthetic RNA. The gal-specific (2). With this probe, we could detect all of the gal mRNA species mRNAs were amplified with a specific PCR primer and primer except mK2 (i.e., mE1, mE2, mT1, mK1, and mM1). The A. The 3′ ends were visualized and located by a primer extension amounts of mK1 and mM1 in WT/pSpot 42 cells were slightly reaction followed by a DNA sequencing gel electrophoresis. We performed the 3′ RACE assay on total RNA isolated from WT and the spf mutant cells (MG1655 Δspf). The results showed that the 3′ ends of mT1 from WT cells are located from +2124 to +2169 (Fig. 6A). However, in spf mutant cells, the 3′ ends of mT1 were missing or severely down-regulated compared with those in WT cells (Fig. 6A). These results demonstrate that without Spot 42, 3′ end generation of mT1 is impaired, suggesting that Spot 42 has a pivotal role in the generation of mT1 3′ ends and is possibly needed for transcription termination at the galT–galK junction.

Hfq Has an Important Role in Rho-Mediated Transcription Termination at the galT–galK Junction. Hfq, a homo-hexameric protein that is known to facilitate base-pairing interactions between sRNA and its target mRNA (3), binds to the 3′ end of Spot 42. The Hfq-Spot 42 complex binds to the 5′ region around the SD sequence of the galK transcript (12). Thus, Hfq itself may be a positive factor for transcription termination at the galK-galT junction. Before we test the role of Hfq in transcription termination at the galK-galT junction, we assayed Rho in transcription termination at the galT–galK junction. We performed 3′ RACE and visualized the 3′ ends of mT1 in the rho mutant strain HME60 (2). The results showed that the 3′ ends of mT1 were decreased in HME60 cells (Fig. 6B), confirming that the 3′ ends of mT1 are generated by Rho-mediated transcription termination. Furthermore, we performed 3′ RACE and visualized the 3′ ends of mT1 in WT cells that have been treated with the Rho-inhibitor, bicyclomycin (22), for 10 min. The ′ Fig. 6. The 3′-ends of mT1 from WT and WT Δspf are shown (A). The 3′ ends results showed that the mT1 3 ends were further reduced (Fig. 6B), ′ demonstrating that the 3′ ends of mT1 are indeed generated by were visualized using 3 RACE followed by DNA sequencing gel electrophoresis. The numbers indicate the position of each end from the tran- Rho-mediated transcription termination. + ′ scription initiation site of the P1 promoter ( 1). DNA sequencing ladders We assayed the 3 ends of mT1 in a hfq mutant strain were used as molecular standards. (B)The3′ ends of mT1 are assayed in WT (MG1655Δhfq) and observed that the 3′ ends of mT1 at from cells, WT cells treated 10 min with bicyclomycin (BCM), MG1655Δhfq,and +2124 to +2129 were reduced to 50%, and at from +2165 to + HME60 (rho mutant) cells.

7584 | www.pnas.org/cgi/doi/10.1073/pnas.1424683112 Wang et al. Downloaded by guest on September 30, 2021 lowered production of GalK by Spot 42, in addition to the translational inhibition of galK transcripts suggested previously (11), might be caused by the degradation of mK2 and the enhancement of transcription termination at the galT–galK junction. We have observed that there is a region of ∼100 nucleotides in the nontemplate DNA strand (same sequence as mRNA) in which the cytosine content is higher and the guanine content is lower than in the surrounding region. This region, called the CG bubble (23), is present in every cistron junction in the gal operon, with the exception of the galK–galM junction, as well as the end of the galM gene (1). The CG bubble also appears in all of the known Rho-dependent transcription termination sites at the trp t′, tyrT, bacteriophage f1 IV, lambda tR1, tnaC, hisG2148 mutant, and ilV468 mutant genes analyzed (24) (Fig. S2). In all these and the gal genes, Rho-dependent transcription termination occurs in or after a CG bubble. Thus, the CG bubble region on mRNA appears to serve as a Rho-binding site. An interesting observation is that at the galT–galK junction, Fig. 7. (A) The 3′ ends of mT1 in WT cells harboring plasmid pBRlac, a there are three regions or sequences that operate with different control plasmid for pSpot42, and plasmid pSpot42. The mT1 3′ ends were functions (Fig. 8A). One is the CG bubble, which appears from assayed and visualized using 3′ RACE. (B) Northern blot analysis with the T probe in WT cells harboring the plasmid pBRlac or pSpot42. The RNAs were visualized using the P32-labeled T probe. Note that the T probe detects mE1, mE2, mT1, mK1, and mM1 only.

less than those of the corresponding mRNAs in WT (Fig. 7B), demonstrating again that Spot 42 down-regulates galK by a mechanism that is different from target mRNA degradation. Most surprisingly, the amount of mT1 in WT/pSpot42 was increased fourfold compared with that of mT1 in WT (Fig. 7B), suggesting that Spot 42 could increase galT expression. Taken together, these results demonstrate that Spot 42 down-regulates the generation of mK1 and mM1 and up-regulates the generation of mT1. Considering the location of the Spot 42 binding site in mT1, at the 3′ end of mT1, one of the possible mechanisms to explain the increase in mT1 by Spot 42 is that Spot 42 somehow causes enhanced transcription termination at the galT-K junction (see Discussion). Discussion Results from this study suggest that Spot 42 regulates galK ex-

pression at two levels: target RNA degradation and transcription MICROBIOLOGY termination. Both result in the down-regulation of galK. How- ever, the alleviation of the Spot 42-mediated galK down-regulation in the late exponential growth period does not seem to be solely responsible for galK > galT, because in an operon in which transcription level shows polarity resulting from transcription termination, anything that generates higher downstream levels of gene transcripts than upstream must be affecting degradation rate. We propose that mK2 has an intrinsic longer half-life than other gal mRNA species. During the early exponential growth Fig. 8. (A) The gal mRNA is represented by the thick horizontal line, on period (up to an A600 value of 0.2), when Spot 42 concentration in the cell is high, Spot 42 might successfully compete with the which the ribosome binding site (SD sequence) for galK, Spot 42 binding site, ′ ribosome in binding to mK2. Thus, degradation of mK2 prevails, and the 3 ends of mT1 are shown. The Rho-binding site (CG bubble) is schematically shown over the computer-generated cytosine (C, gray line) which would result in type 2 polarity, in which the amount of galK and guanine (G, dark line) contents over the gal mRNA region from +1990 to is less than that of galT (Fig. 2). Although the effect is not as +2200. Note that the Spot 42 binding site overlaps with the region of the CG pronounced as in mK2 degradation, enhancement of transcrip- bubble. (B) A hypothetical model for transcription termination at the galT– tion termination by Spot 42 at the galT–galK junction would galK junction. By the time RNA polymerase passes the stop codon of galT, supplement the down-regulation of galK expression as well. the first ribosome that is coupled to the RNA polymerase would reach the stop codon. The binding site of Spot 42 would be located between the first However, during late exponential growth (from A600 values of 0.2–1.86), when there is less Spot 42, ribosomes would win the ribosome and the RNA polymerase on the newly transcribed RNA. Binding of competition for mK2 binding, leading to less mK2 degradation Spot 42, presumably with Hfq, should cause disassembly of the whole structure, including Spot 42 itself, to expose the putative Rho-binding site by Spot 42 and increased translation of galK. In addition, in this (CG bubble). Rho would eventually catch up with the RNA polymerase now growth period, increased transcription of galK, and possibly the transcribing downstream DNA and terminate transcription. The thin arrows longer mK2 half-life, would result, leading to type 1 polarity, indicate the direction of RNA polymerase. The thick arrow on the transcript where the amount of galK is greater than that of galT. Thus, the indicates the direction of Rho.

Wang et al. PNAS | June 16, 2015 | vol. 112 | no. 24 | 7585 Downloaded by guest on September 30, 2021 +2075 to +2175; the second is the SD sequence for galK, which the first ribosome terminates translation at the stop codon of extends from +2090 to +2096; and the third is the binding site galT. The disassembly of the first ribosome would reveal the for Spot 42, which spans from +2084 to +2158 (Fig. 8A). It is putative Rho-binding site. Rho-mediated transcription termina- difficult to conceive how the binding of Spot 42 at the galT–galK tion occurs at a point downstream of galT. Now, the Spot 42-Hfq junction enhances Rho-termination, because the binding site of complex binds to its binding site and stops exoribonucleolytic Spot 42 overlaps with the CG bubble to which Rho likely binds to processing at the region from +2165 to +2169, which is at the 5′ terminate transcription. Spot 42 binding should interfere with end of the bound Spot 42, and promotes endoribonucleolytic Rho binding; therefore, overexpression of Spot 42 should reduce cleavage at the region from +2124 to +2129, which is in the Rho-termination at the galT–galK junction, rather than enhanc- middle of the Spot 42 binding region (Fig. 8A). ChiX sRNA- ing it. We wish to propose a molecular mechanism that possibly assisted Rho-mediated termination has been demonstrated in explains the enigmatic result that Spot 42 enhances Rho-termi- the chiPQ locus of Salmonella (6). However, the molecular nation at the cistron junction, instead of reducing it. This model mechanism involved in enhancing Rho-mediated termination is is schematically presented in Fig. 8. It was proposed, and shown different. In the gal operon, sRNA binding occurs in a cistron recently, that transcription and translation are tightly coupled in junction, and in the chiPQ operon, sRNA binding occurs at the E. coli (25, 26). If the first ribosome coupled to the RNA poly- beginning of the first gene. Thus, the consequence of sRNA galT merase reaches the stop codon of the gene, the only way for binding appears to be different between the two systems. For Rho to bind to the putative Rho binding site (the CG bubble these reasons, we believe that the role of Spot 42-Hfq in Rho- region) is when the first ribosome terminates translation and is termination at the galK-galT junction is unique and that this disassembled completely; this disassembly of the ribosomal report is the first demonstration of its kind. complex would leave the CG bubble free of ribosomes and available for Rho binding (Fig. 8). We propose that Spot 42 Materials and Methods binding to its cognate site at the galT–galK junction enhances the disassembly of the ribosome at the stop codon of galT. Possibly, Chromosomal deletion of the cya and spf genes from the MG1655 strain was ′ carried out by λ red-mediated recombination, as described previously (30). Hfq now bound at the 3 end of Spot 42 might help the disas- The plasmids pBRplac and pSpot 42 were generous gifts from Beisel CL (13). sembly of the ribosome. Otherwise, translation would continue In all experiments, cells were grown at 37 °C in LB medium (10 g tryptone, to galK (14); in that case, there would be no transcription ter- 5 g yeast extract, and 10 g NaCl per liter of water) supplemented with 0.5% mination. Rho would traverse RNA to catch up to the RNA (wt/vol) galactose. For strains containing the pBRplac or pSpot 42 plasmids, polymerase now transcribing downstream DNA, and eventually ampicillin was added at a final concentration of 100 μg/mL RT-qPCR, 3′ RACE, terminate transcription (27, 28). However, the actual 3′ end of and Northern blot analysis were conducted essentially as described pre- mT1 is within the Rho binding site in the CG bubble region (Fig. viously (1, 2), with modifications as indicated in the figure legends. Primers 8A). It is likely that Rho-mediated transcription termination used in this study are listed in Table S2. occurs downstream, as described earlier, but the 3′ end of mT1 (shown in Fig. 6) is generated by exoribonucleolytic processing of ACKNOWLEDGMENTS. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, the terminated transcript, as demonstrated in the case of Rho- funded by the Ministry of Education, Science, and Technology (2013007271). termination at the end of the trp operon (29). An alternative This research was also supported by a grant from Chungnam National model is that transcription and translation are decoupled when University (2014-785-01).

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