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

Xun Wang,a Sang Chun Ji,a* Sang Hoon Yun,a* Heung Jin Jeon,a Si Wouk Kim,b Heon M. Lima

Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, Republic of Koreaa; Department ofEnvironmental Engineering, Pioneer Research Center for Controlling of Harmful Algal Blooming, Chosun University, Gwangju, Republic of Koreab

The 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, andmM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural po-larity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicatedthat 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 transcriptioninitiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 arepartially 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 be-come mE2. These results also suggested that galK expression could be regulated through mK2 production independent fromnatural polarity.

Polycistronic operons in bacteria show a differential expressionof the constituent cistrons (1). A Northern blot analysis

showed that there are 6 different species of mRNA specific to thegalactose operon in wild-type E. coli cells grown exponentially inthe presence of galactose (2). Five of the 6 mRNA species, mE1,mE2, mT1, mK1, and mM1, have their 5= ends at the transcriptioninitiation region, and their 3= ends at 5 different locations withinthe operon, four of which (all but mE2) are at the ends of the galE,galT, galK, and galM cistrons, respectively (Fig. 1A). There is onedistinct mRNA species, designated mK2, that has 5= ends not atthe promoter region but at the middle of galT. The existence ofthese mRNA species automatically establishes a gradient of geneexpression, higher in the promoter-proximal region and lower inthe promoter-distal region, which has been referred to as “naturalpolarity” (3). Natural polarity is intrinsically different from whathas been known as polarity that is caused by a mutation (4), be-cause it can be observed in cells harboring the wild-type operon (2,5–9). The term “polarity” refers to the phenomenon in which amutation in one gene of an operon decreases the expression of thesubsequent genes of the operon. The cause for polarity is wellestablished. The cessation of translation by a nonsense mutationuncouples transcription from translation, allowing the transcrip-tion termination factor, Rho, to bind to the nascent RNA andterminate transcription at the next available termination signal.This Rho-mediated transcription termination leaves the rest ofthe operon untranscribed, creating polarity (3, 4, 10, 11).

In our previous report (2), we could not determine the specificcause of natural polarity in the gal operon. In this study, we askedwhether the natural polarity in gal is caused by (i) mRNA process-ing of a long transcript by endoribonucleases, (ii) differential de-cay rate of the different gal mRNAs, (iii) intrinsic DNA sequenceof the gal operon, or (iv) intraoperonic transcription termination.Results from a quantitative measurement of the ends of tran-

scripts from a series of 3=-end deletion constructs of the gal operonindicated that transcription termination at the end of each cistronis the primary cause of natural polarity.

Expression of the galK gene in the gal operon is regulated bySpot 42, a small RNA (sRNA) that belongs to a group of noncod-ing RNA of a predominant size range of 50 to 250 nucleotides. ThesRNA controls gene expression through sequence-specific bind-ing to the target mRNA, causing target RNA degradation or trans-lation inhibition (12). Spot 42 decreases GalK production bybinding to the ribosome binding site of the galK transcript (13)and overproduction of Spot 42 from a plasmid decreases galKtranscript (14). Among the 6 gal-specific mRNAs, mK1, mK2, andmM1 have the binding sites for Spot 42. We hypothesized that thetarget mRNA for Spot 42 is mK2, because only mK2 has the Spot42 binding site near the 5= end. Spot 42 may not be able to bindmK1 and mM1 due to the translating ribosomes. During the ex-periments designed to answer whether the cause of natural polar-ity resides at mRNA processing, we found that the 5= end of mK2is produced by a RNase P-mediated cleavage of a long gal tran-script. In this study, we also asked (i) how mK2 is produced and(ii) how the production of mK2 is related to the establishment ofnatural polarity.

Received 15 February 2014 Accepted 26 April 2014

Published ahead of print 2 May 2014

Address correspondence to Heon M. Lim, hmlim@cnu.ac.kr.

* Present address: Sang Chun Ji, Department of Clinical Pharmacology andTherapeutics, Seoul National University College of Medicine and Hospital, Seoul,South Korea; Sang Hoon Yun, Alteogen Inc., Daejeon, Republic of Korea.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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MATERIALS AND METHODSBacterial strains and growth conditions. Strain MG1655 was used as thewild-type (WT) strain in this study. Chromosomal deletion strains of theentire gal operon were generated by deleting the corresponding gene(s)from MG1655 using � red-mediated recombination (15). The primersused are listed in Table S1 at http://cnu.ac.kr/~hmlim/. The MG1655�cyastrain was described previously. The RNase E temperature-sensitive mu-tant GW20 (W3110 zce-726::Tn10 rne-1), RNase G-defective strain GW11(W3110 zce-726::Tn10 rng::cat), and strain GW10 (W3110 zce-726::Tn10), from which GW11 and GW20 were derived, were provided by H.Aiba (Nagoya University, Japan). An RNase P temperature-sensitive mu-tant, NHY322 [�(proBlac) ara gyrA thi zic-501::Tn10 rnpA49], and strainNHY312 [�(proBlac) ara gyrA thi zic-501::Tn10 rnpA�], from whichNHY322 was derived, RNase III mutant strain SDF205 (W3110 rnc105TD1-17::Tn10), and strain SDF204 (W3110 TD1-17::Tn10), from whichSDF205 was derived, were provided by Y. H. Lee (KAIST, South Korea).HME60 (W3110 rho::bla; nine amino acids of rho at the C terminus werereplaced by bla) was provided by D. Court (NIH, USA) (16, 17). Exceptfor GW20, GW10, NHY322, and NHY312, which were cultured at 30°Covernight, cells were grown at 37°C in LB medium (10 g tryptone, 5 g yeastextract, and 10 g NaCl per liter of water) supplemented with 0.5% (wt/vol)galactose. After a 1/100 dilution in fresh medium, cells were grown at 30°Cfor 1 h and then incubated at a nonpermissive temperature (44°C) for anadditional hour before harvest and further processing. Nine bases existbetween the stop codon of galE and the start codon of galT, while threebases exist between galT and galK. The stop codon of galK overlaps thestart codon of galM.

RNA preparation. Equal numbers of cells (about 2 � 108) were har-vested at an A600 of 0.6 and resuspended in 50 �l protoplasting buffer (15mM Tris-HCl, pH 8.0, 0.45 M sucrose, and 8 mM EDTA). Five microlitersof lysozyme (50 mg ml�1) was added, and then the sample was incubatedfor 5 min at 25°C. A phenolic detergent (1 ml TRI Reagent; MolecularResearch Center, USA) was added, and the mixture was vortexed for 10 sbefore incubation for 5 min at 25°C. Chloroform (200 �l; Sigma-Aldrich,USA) was added to the mixture, which was vortexed vigorously for 20 s

and then incubated for 10 min at 25°C. The resulting mixture was centri-fuged at 10,000 � g for 15 min at 4°C. The aqueous phase (500 �l) wastransferred to a new tube, mixed with 500 �l of isopropanol (Sigma), andthen incubated for 10 min at 25°C. RNA was collected by centrifugation at10,000 � g for 15 min at 4°C and washed with 1 ml of 75% cold ethanol.The precipitated RNA was dissolved in 50 �l of RNA storage buffer (Am-bion, USA). RNA concentration was determined by measuring the absor-bance at 260 nm using a NanoDrop spectrophotometer (Thermo FisherScientific, USA).

qRT-PCR. For quantitative real-time RT-PCR (qRT-PCR), genomicor plasmid DNA in the reaction mixture was removed with Turbo DNA-free (Ambion) according to the manufacturer’s recommendations. Re-verse transcription was performed with the following method: 2.0 �g oftotal RNA was incubated at 37°C for 2 h in a 20-�l reaction volumecontaining 4 U of Omniscript reverse transcriptase (Qiagen, Germany),0.5 mM each deoxynucleoside triphosphate (dNTP), 10 �M random hex-amer primer (TaKaRa, Japan), and 10 U of rRNasin (Promega, USA).PCR primer sets used in qRT-PCR are listed in Table S1 at http://cnu.ac.kr/~hmlim/. qRT-PCR was performed in 10-�l reaction mixtures con-taining 5 �l of iQ SYBR green Supermix (Bio-Rad, USA), 3 �l of nuclease-free water, 0.5 �l each of forward primer (10 mM) and reverse primer (10mM), and 1 �l of the cDNA template under the following conditions: aninitial denaturation step at 94°C for 3 min, and then 40 cycles of 15 s ofdenaturation at 94°C, 20 s of hybridization at 60°C, and 15 s of elongationat 72°C (CFX96; Bio-Rad). The results from each strain were normalizedagainst those for the rrsB gene coding for 16S rRNA.

3= RACE and 5= RACE assay. Total RNA was extracted as describedabove. For the 3= RACE assay, RNA ligation was performed at 37°C for 3h in a 15-�l reaction volume containing 2.5 �g of total RNA, 2 nM syn-thetic RNA oligomer possessing a 5=-phosphate and 3=-inverted de-oxythymidine (27-mer; Dharmacon, USA), 5 U of T4 RNA ligase, and 10U of rRNasin. The RNA ligation reaction was applied to a G-50 column.One microgram of RNA (eluted from the G-50 column) was reverse tran-scribed at 37°C for 2 h in a 20-�l reaction volume containing 4 U ofOmniscript reverse transcriptase, 0.5 mM each dNTP, 0.4 �M 3RP primercomplementary to the RNA oligomer (see Table S1 at http://cnu.ac.kr/~hmlim/), and 10 U of rRNasin. A 2-�l sample of this reaction was usedas the template for PCR amplification of the gal cDNA with gene-specificprimers and the 3RP primer (see Table S1 at http://cnu.ac.kr/~hmlim/)using HotStar Taq DNA polymerase (Qiagen, Germany). To assay the 3=ends of the gal mRNAs, the amplified cDNA was purified and used as atemplate for a primer extension reaction performed in a volume of 20 �lwith a 32P-labeled primer (complementary to different regions of the galoperon mRNA; see Fig. 4) and 1 U of Taq polymerase (Qiagen, Germany).The reaction products were resolved on an 8% polyacrylamide urea se-quencing gel, and the radioactive bands were visualized after exposure toX-ray film. The 3= ends of mE2 were not detected in the 3= rapid amplifi-cation of cDNA ends (RACE) assay. We believe that the 3= ends thatappear to be the result of RNA processing did not ligate to the RNAaptamer during the ligation reaction in the 3= RACE assay.

The procedure for 5= RACE was almost the same as that for the3=RACE assay, with a little modification. The 5S rRNA was used as anRNA aptamer for ligation instead of synthetic RNA oligomer. For thereverse transcription, a final concentration of 10 �M random primer(hexamer; TaKaRa, Japan) was used. PCR amplification of the gal cDNAwas performed with a forward primer complementary to the 3= end of 5SrRNA and a reverse primer specific to the galK region (see Table S1 athttp://cnu.ac.kr/~hmlim/).

Measurement of gal mRNA decay kinetics. MG1655 cells were cul-tured until early-log-phase growth was achieved (optical density [OD] of0.6). Rifampin was added to the culture to a final concentration of 500 �gml�1, and cells were harvested at 0, 2, 4, 6, and 8 min. Harvested cells (2 �108 at each time point) were mixed immediately with 10% buffer-satu-rated phenol in ethanol (1/10 volume) and chilled rapidly on ice. RNA

FIG 1 gal-specific mRNAs and two different types of mCONGRAD. (A) Aschematic representation of the galactose operon in E. coli. �1 indicates thetranscription initiation site from the P1 promoter (P1). Numbers indicate thepositions of the stop codons for the cistrons relative to �1. The five gal-specificmRNAs that differ only in their 3= ends are shown. Double arrows indicate thelocation of the primers used for qRT-PCR to measure mCONGRAD. The sixthgal mRNA, mK2, encodes galactokinase and has a 5= end different from that ofother gal mRNAs. The stem-and-loop structure at the end of the operon isshown. (B) The type 1 mCONGRAD measured from WT MG1655 cells grownexponentially in LB with 0.5% galactose using the qRT-PCR primers shown inpanel A. (C) The type 2 mCONGRAD measured from MG1655�cya cells.

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preparation, cDNA synthesis, and real-time PCR were performed as de-scribed above.

In vitro transcription assay. The pHL1277 plasmid was used as aDNA template for in vitro transcription. pHL1277 was obtained by clon-ing the entire galactose operon into the pCC1BAC plasmid. The in vitrotranscription reaction was performed using Escherichia coli E�70 (Epicen-tre) according to the manufacturer’s instructions. Briefly, DNA template(2 nM) was incubated at 37°C for 5 min in reaction buffer (20 mM Tris-acetate, pH 7.8, 10 mM magnesium acetate, 200 mM potassium gluta-mate, 1 mM ATP, and 1 mM dithiothreitol [DTT] [18]) containing 2 UE�70 and 40 U rRNasin (Promega) in a 47.5-�l reaction. The reaction wasinitiated by adding 2.5 �l NTP mix (final concentration, 0.1 mM eachNTP) to the mixture. After 30 min, the reaction was terminated by phe-nol-chloroform extraction, and then 30 �l of supernatant was purifiedusing a G-50 column. Purified RNA was then used for qRT-PCR or 3=RACE assays as described above.

Plasmid construction. The pHL1277 plasmid was constructed by in-serting the galactose operon (from �75 to �4333) between the EcoRI andBamHI sites of pCC1BAC (Epicentre Biotechnologies, USA). PCR prim-ers used to amplify the galactose operon from genomic DNA are listed inTable S1 at http://cnu.ac.kr/~hmlim/. Sequences corresponding toBrevibacterium albidum tRNAarg from pCAT-tRNAArg (19) were ampli-fied by PCR using primer sequences shown in Table S1 at http://cnu.ac.kr/~hmlim/, digested with HindIII, and then inserted immediately up-stream of the cat gene in pKK232-8 (GE Healthcare, USA) to create thepHL1141 plasmid. DNA fragments containing different portions of the galoperon, from nucleotide position �73 to various positions downstream,were obtained via PCR amplification of the genomic DNA with the corre-sponding primer pairs (see Table S1 at http://cnu.ac.kr/~hmlim/). The result-ing PCR fragments were digested with BamHI and SalI and then ligated topHL1141 to generate pHL1142, pHL1143, pHL1144, pHL1145, andpHL1146.

Northern blot analysis. Total RNA was isolated as described above. Aspecific amount (depending on the experiment; see below) of total RNA(with 1 mg/ml ethidium bromide) was resolved by 1.2% (wt/vol) formal-dehyde-agarose gel electrophoresis at 5 V/cm for 4 h. After electrophore-sis, RNA integrity was assessed under UV light and the RNA was trans-ferred overnight to a positively charged nylon membrane (Ambion, USA)using a downward transfer system (TurboBlotter; Whatman, UnitedKingdom) and then fixed to a nylon membrane by baking at 80°C for 1 h.The Northern probe was prepared as follows. First, a 500-bp DNA frag-ment in the galK region (from �2103 to �2603) was prepared by PCRusing primers indicated in Table S1 at http://cnu.ac.kr/~hmlim/. TheRNA probe (generated from the 500-bp DNA fragment by in vitro tran-scription) then was labeled with digoxin according to the manufacturer’sprotocol (DIG Northern starter kit; Roche, Switzerland). The transferredRNA was hybridized with the RNA probe (100 ng/ml) at 68°C for 8 h anddetected immunologically using the anti-digoxin antibody by chemilumi-nescence.

RESULTSThe 6 mRNAs of the gal operon and their measurement. Wehave demonstrated in our previous report (2) with quantitativereal-time RT-PCR (qRT-PCR) using primers specific to each cis-tron of the gal operon (Fig. 1A) that expression of galE is greaterthan that of galT, and that galM has the lowest level of expressionof the four cistrons (Fig. 1B). The level of galK transcript, due to itsmore distal location from the promoter, was expected to be lessthan that of galT; however, galK expression actually was found tobe greater than that of galT. This exception to the transcriptiongradient may be due to the presence of another gal-specific mRNAspecies, mK2 (Fig. 1A and B), which depends on cyclic AMP(cAMP). We termed this phenomenon, which provides naturalpolarity at the level of transcription, the mRNA concentration

gradient (mCONGRAD). Thus, mCONGRAD measured in theWT shows more galK than galT, and we termed this type 1. How-ever, when mCONGRAD is measured in a cya mutant strain, galTis greater than galK, and we termed this type 2 (Fig. 1C). Through-out this study, unless otherwise noted, mCONGRAD was mea-sured in cells grown in LB containing 0.5% galactose. Moreover,total RNA was isolated from 2 � 108 cells during early-log-phasegrowth (OD600 of 0.6). The same amount of total RNA (2 �g) wasused for qRT-PCR.

RNase E, III, and G are not involved in 3=-end generation ofthe gal mRNAs. To better understand mCONGRAD and itscauses, we investigated several possible factors that could influencehow the 3=ends of the gal mRNAs are generated. First, we investigatedthe effects of processing full-length mRNA (mM1) into differentsizes. To accomplish this, we measured mCONGRAD in E. colistrains carrying mutations in endoribonucleases known to be in-volved in RNA processing. WT MG1655 cells exhibited a typicalmCONGRAD status (type 1) with the relative amount of galT,galK, and galM transcripts measured as 0.5, 0.65, and 0.07 of thegalE transcript (Fig. 1B). Results from the endoribonuclease mu-tant strains showed that the mCONGRAD status at a nonpermis-sive temperature (44°C) for the endonuclease RNase E tempera-ture-sensitive strain (lacking rne) was similar to that of theisogenic strain (rne�) from which the mutant strain was derived(Fig. 2). The mCONGRADs in the G-negative (rng::cat) andRNase III (rnc105) strains were also identical to those of the cor-responding WT strains (rng� and rnc�) (Fig. 2). Although thetotal amount of transcript in the RNase E and RNase III strainswas 0.34 and 0.26, respectively, relative to the corresponding WTstrain, the expression ratio of genes to galE did not change (Table1). These data suggest that RNA processing by RNase E, III, or G isnot involved in generation of the 3= ends of the gal mRNAs.

RNase P-mediated cleavage of a pre-mK2 mRNA could yield5= ends of mK2 and possibly 3= ends of mE2. Interestingly,mCONGRAD in the temperature-sensitive RNase P strain(rnpA49) at the nonpermissive temperature (44°C) appeared thesame as that observed in a cAMP-deficient (MG1655 �cya) strainlacking mK2 (Fig. 2 and Table 1). In this type 2 mCONGRADevent, the amount of galK was less than that of galT, yielding a

FIG 2 mCONGRAD measurements of gal mRNAs in RNase mutant strains.GW10 (rne�, RNase E control) cells were grown at 30°C for 1 h and thenshifted to 44°C for 1 h. Strains used were GW20 (rne mutant; RNase E tem-perature sensitive), GW10 (rng�; RNase G control), GW11 (rng::cat; RNase Gdownmutation), SDF204 (rnc�; RNase III control), SDF205 (rnc105; RNase IIIdownmutation), NHY312 (rnpA�; RNase P control), NHY322 (rnpA49;RNase P temperature sensitive). Independent measurements were performedat least three times. The amount of transcript relative to that of galE in eachcontrol is presented.

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linear gradient of mRNA concentration. This linear gradient inthe absence of RNase P activity implies its potential involvementin mK2 production. Therefore, we expected that there would beno mK2 mRNA in the RNase P mutant strain. We visualized mK1,mK2, and mM1 by Northern blotting (Fig. 3) and found that therewas no discrete band of mK2 in the RNase P mutant at the non-permissive temperature.

It was envisioned that the 5= ends of mK2 are generated byRNase P-mediated endonucleolytic digestion of a pre-mK2mRNA. We searched the 5= ends of mK2 by using the 5= RACEassay. Briefly, 5= ends of total RNA were ligated to the 3= end of the5S rRNA, which is abundant in total RNA preparations. Reversetranscription was performed using a random primer. PCR thenwas performed with a primer specific to the 3= end of the 5S rRNAand a gal-specific primer that binds to the beginning of galK (2).This PCR would amplify the cDNA whose 5= ends are roughlybetween �1500 and �2100, where 5= ends of mK2 might reside(Fig. 1A). The amplified 5= ends were detected using a primerextension reaction with 5 different radioactive primers. We iden-tified two 5= ends at �1764 and �1777 that could be observed ina WT control strain of the RNase P mutant but disappeared fromthe RNase P temperature-sensitive mutant strain at the nonper-missive temperature (Fig. 4). Based on these results, we concludedthat the 5= ends of mK2 are generated by RNase P digestion of apre-mK2 at �1764 and �1777. By simple logic based on the lo-cation of the 3= ends of mE2, which are at the middle of galT andclose to the 5= ends of mK2, we suppose that the 5= portion of the

gal mRNA generated by the RNase P digestion of the pre-mK2would become mE2.

Neither RNA decay rates nor gene sequence is involved inmCONGRAD formation. We next addressed whether differentdecay rates of the gal mRNAs could account for the generation ofdifferent species of the gal mRNAs. mCONGRAD was measuredin WT cells at 0, 2, 4, and 8 min after addition of rifampin, aninhibitor of bacterial RNA polymerase (RNAP), to the culture.The relative amount of transcript representing each cistron de-creased after rifampin treatment. When changes in the amount ofgalE, galT, galK, and galM transcripts were plotted over time, itbecame evident that transcripts representing each cistron de-graded at nearly the same rates (Fig. 5). The level of transcriptcorresponding to each cistron is the sum of different mRNA spe-cies (e.g., the amount of galE is the sum of mE1, mE2, mT1, mK1,and mM1). Therefore, these results showing that the gal tran-scripts undergo similar decay rates while maintaining the samemCONGRAD suggests that the general RNA degradation processdoes not lead to the generation of the 3= ends of the gal mRNAs.

We then investigated whether the DNA sequence itself, or aproperty intrinsic to the DNA sequence at the end of each cistron,could terminate transcription. We measured the mCONGRAD oftranscripts derived from in vitro transcription of the entire galoperon cloned into a low-copy-number plasmid, pHL1277 (seeMaterials and Methods). Our results demonstrate that theamount of transcript representing the first three cistrons, namely,galE, galT, and galK, were identical, and that the level of galMtranscript was almost half that of the others (Fig. 6). The data thatgalM transcription is half that of galK suggest that RNAP alone(without any protein factors) could terminate transcription at theend of galK and that mK1 could be generated by transcription

TABLE 1 Expression of the gal genes relative to the first gene, galE

Strain Phenotype

Expression ofa:

galT galK galM

MG1655 Wild type 0.50 0.14 0.65 0.09 0.07 0.02MG1655�cya Cyclase deletion 0.43 0.04 0.39 0.03 0.07 0.01GW10 (rne� rng�) Wild type 0.60 0.16 0.82 0.06 0.08 0.01GW20 (GW10

mutated in rne)RNase E tsb 0.59 0.06 0.80 0.09 0.10 0.02

GW11 (GW10rng::cat)

RNase G downmutation

0.49 0.07 0.80 0.09 0.07 0.02

SDF204 (rnc�) Wild type 0.56 0.09 0.63 0.15 0.09 0.03SDF205 (SDF204

rnc105)RNase III down

mutation0.46 0.20 0.55 0.13 0.07 0.06

NHY312 (rnpA�) Wild type 0.51 0.07 0.79 0.28 0.07 0.02NHY322 (NHY312

rnpA49)RNase P ts 0.46 0.08 0.35 0.10 0.06 0.01

a Results are given with standard deviations.b ts, temperature sensitive.

FIG 3 Northern analyses of mK2, mK1, and mM1 in the RNase P mutantstrain. rnpA�, RNase P control strain; rnpA49, RNase P temperature-sensitivestrain. Cells were grown at 30°C for 1 h and then shifted to 44°C for 1 h.

FIG 4 5= Ends of mK2 visualized by 5= RACE assay of the rnpA� RNase Pcontrol strain and the RNase P temperature-sensitive strain (rnpA49). Cellswere grown at 30°C for 1 h and then shifted to 44°C for 1 h. Numbersindicate the positions of the 5= ends of mK2 relative to the transcriptioninitiation at �1.

FIG 5 Decay kinetics of the gal transcripts. Relative amounts of the galmRNAs to that of galE at time zero are presented. The relative amount ofmRNA was plotted against time to determine the decay rate.

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termination at the end of galK. The data that the amount of galE,galT, and galK is the same suggest that an in vivo factor(s) existsthat governs the generation of the 3= end of mE1 and mT1. Theamount of galK is greater than that of galM in all of the RNasemutants tested in Fig. 2, suggesting that the galK termination isnot the result of RNA processing.

Transcription termination generates 3= ends of the galmRNAs. Because we found that the 3= ends of mE1, mT1, mK1,and mM1 are not generated by differential endoribonuclease pro-cessing or mRNA decay rates, we investigated intraoperonic tran-scription termination in the gal operon. Based on the locations ofthe 3= ends of mE1, mT1, mK1, and mM1 in the gal operon shownin Fig. 1A, we anticipated transcription termination at the end ofeach cistron. For this, we constructed a series of gal operon dele-tion mutants and cloned the deletions in a high-copy-numberplasmid, pHL1141. As diagrammed in Fig. 7A, each plasmid hasthe same 5= portion of the gal operon, including both the P1 andP2 promoters and operators, but different 3= ends. The plasmidpHL1142 has a portion of the gal operon from �73 to �1031,pHL1143 has up to �1699, pHL1144 has up to �3087, pHL1145has up to �3791, and pHL1146 has the entire gal operon, from�73 to �4344. The argX gene coding for tRNAarg from Brevibac-terium albidum is cloned at the end of each gal deletion. If tran-scription termination occurs at the end of each cistron, theamount of the tRNAarg transcribed from these plasmids will be thegreatest in the longest deletion plasmid, pHL1142, and smallest inthe shortest deletion plasmid, pHL1146.

We performed qRT-PCR to measure the amount of tRNAarg

expressed in MG1655 cells harboring the plasmids described above.Our results demonstrated the formation of a gradient of tRNAarg

expression, with the longest deletion construct (pHL1142) showingthe greatest tRNAarg expression and the shortest deletion con-struct (pHL1146) exhibiting the least (Fig. 7B). There was a grad-ual decrease in tRNAarg levels in cells harboring the intermittentconstructs. These data, along with the location of the 3= ends ofmE1, mT1, mK1, and mM1 in the gal operon, suggest the likeli-hood of transcription termination at the end of each cistron. Us-ing the equation termination frequency 1 � readthrough/up-stream transcripts (20), we were able to calculate the transcriptiontermination frequency occurring at different sites in the operon(Table 2). These data suggested that these terminations are sto-chastic. Each termination occurs with a finite efficiency of less than100%. The half-life of the tRNAarg transcripts from the various con-structs was measured and found to be similar in all cells, with anaverage of 1.3 min (see Fig. S2 at http://cnu.ac.kr/~hmlim/). Thesedata indicate that intraoperonic transcription termination generatesthe 3= ends of mE1, mT1, mK1, and mM1.

The transcription terminations are Rho dependent. We nexttested whether the transcription termination at the end of each

cistron is mediated by the Rho factor. We measured the amount oftRNAarg from the same series of plasmids used above in the Rho-impaired E. coli strain, HME60. The mutant rho gene rho-15 (16,17) in the HME60 strain has been selected as a nonfunctionaltranscription terminator, and the corresponding mutant Rhoprotein differs from that of the WT by nine amino acids at its Cterminus (D. Court, NIH, USA, personal communication). Ourresults showed that, contrary to WT cells, there was little variationin tRNAarg levels in HME60 cells harboring pHL1142, pHL1143,and pHL1144 (Fig. 7C), suggesting that transcription terminationdid not occur at the end of galE and galT in the rho-15 mutant.Therefore, transcription termination at the end of galE and galT

FIG 6 Relative amount of transcript of each gal cistron from in vitro transcrip-tion of the entire gal operon.

FIG 7 In vivo transcription termination assay. Schematic illustration of theseries of plasmids used to measure intraoperonic transcription in vivo. Eachplasmid contains a 5= portion of the gal operon as indicated. For example,pHL1142 has a portion from �73 to �1031. pHL1146 expresses the entire galoperon from �73 to �4344, including the stem-and-loop structure at the endof galM. The 5= portions of the gal operon were cloned in front of the tRNAarg

gene of pHL1141. A chloramphenicol resistance (cat) gene (the determinant)was fused to the 3= end of tRNAarg. (A) The strong Rho-independent rrnBtranscription termination signal (31) was cloned after the cat gene and also infront of the gal promoters to prevent aberrant transcription originating fromplasmid DNA. Thus, transcription initiated from the gal promoters wouldtranscribe the 5= end of the operon DNA, tRNAarg, and cat before terminationat the rrnB unless terminated at the intercistronic transcription terminator.The relative amount of tRNAarg was measured in MG1655 (B) and in HME60(C), the Rho-negative strain. The amount of tRNAarg was measured usingqRT-PCR and is presented relative to that of pHL1142. Data represent at leastthree independent experiments.

TABLE 2 Transcription termination efficiency at the end of each cistron

Cistron

Termination efficiency (%)

MG1655 WT HME60 (rho::bla)

1031–1669 (end of galE) 16 01669–3087 (end of galT) 29 03087–3791 (end of galK) 65 83791–4344 (end of galM) 71 30

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that generates 3= ends of mE1 and mT1, respectively, is Rho de-pendent. Transcription termination efficiency at the end of galKdrops from 65% in the WT to 8% in HME60, while the corre-sponding values at the end of galM are 71% in the WT and 30% inHME60, suggesting that those two terminations are partially Rhodependent (Table 2). Considering that the galM transcript levelwas half that of galK after in vitro transcription of the gal operon(Fig. 6), these results indicate that the DNA sequence itself at theend of galK contains a transcription stop signal (see below). Sim-ilarly, transcription termination still occurs at the end of galMeven in the absence Rho, because the termination efficiency inHME60 cells was 30% (Table 2). A stem-and-loop structure onmRNA was found 6 bp downstream of the galM translation stopcodon, followed by 3 continuous thymine residues. We inter-preted this sequence as a conventional Rho-independent termina-tor. This Rho-independent terminator may explain why 30% ter-mination still occurs in the HME60 strain.

The specific location of the 3= ends of mK2. The 3= ends ofmE1, mE2, mT1, mK1, and mM1 have been identified andmapped (2), but those of mK2 have not been identified. To inves-tigate the exact location of the 3= ends of mK2, we performed the3= RACE assay to visualize all 3= ends of the mRNAs generatedfrom the entire gal operon. Briefly, 3= ends of total RNA wereligated to a synthetic RNA aptamer composed of 27 nucleotides,and reverse transcription was performed using a DNA primercomplementary to the RNA aptamer (Fig. 8A). PCR was thenperformed with the reverse transcription primer and a gal-specific primer, and the amplified 3= ends were detected using aprimer extension reaction with a radioactive primer. With the5 PCR primers and 21 primer extension primers nestedthroughout the operon, we were able to visualize most of the 3=ends of the mRNAs transcribed from the gal operon (Fig. 8A).

The locations of most 3= ends of gal mRNAs concurred with thedata of our previous report (2), except for mE2 and mK1 (seebelow). The 3= ends of mE1 are located 82 to 160 nucleotidesdownstream from the stop codon of galE. Those of mT1 and mM1are located 25 to 70 and 30 to 51 nucleotides downstream from thestop codons of galT and galM, respectively (data now shown).Several bands that appeared to be the 3= ends of mK2 existed atnucleotides �4025 to �4227 toward the end of galM (Fig. 8B).Based on the size of mK2, about 2.2 kb (2), and the location of its5= ends (�1764 and �1777) (Fig. 4), we concluded that these areindeed 3= ends of mK2. When the same 3= RACE assay was per-formed in the rho mutant strain, HME60, we found that the bandsthat appeared as the 3= ends of mK2 disappeared (Fig. 8B, secondlane). These data clearly demonstrated that the 3= ends of mK2 aregenerated by Rho termination at the specific locations shown inFig. 8B.

The specific location of the 3= ends of mK1. The Northern blotof the gal operon clearly showed a discrete band of mE2 and mK1(2). However, the 3= RACE assay performed to generate Fig. 8 didnot detect any bands specific to the 3= end of mE2 and mK1 (datanot shown). We considered the 3= ends detected with the 14, 15,16, 17, and 18 primer extension primers (Fig. 8A) that are de-signed to detect the end of galK to be non-gal-specific signals,because these same bands were detected in the MG1655�galstrain, which lacks the entire gal operon (see Fig. S1 at http://cnu.ac.kr/~hmlim/). The 3= ends of mK1 reported in our previouspublication (Fig. 7 in reference 2) must have been non-gal specific.One possible reason for this is that the 3= ends of mE2 and mK1 are

engaged in further processing or degradation, and because of thatthe 3= ends are not ligation proficient to the RNA aptamer duringthe 3=RACE assay. We reasoned that we would be able to see the 3=ends of mK1 if we perform 3=RACE on RNAs generated by in vitrotranscription of the entire gal operon, because no RNA degrada-tion or processing is possible in in vitro transcription. With the 14,15, 16, 17, and 18 primer extension primers, we found 3= endsfrom �3218 to �3287 that appear to be the putative 3= ends ofmK1 (Fig. 8C). We could not detect any DNA sequence upstreamof the 3= ends that could form a stem-and-loop structure onmRNA that appears as a signal for intrinsic transcription termina-tion (21). Taking these findings and the interpretation from Fig. 6that there is in vitro transcription termination at the end of galK, itis likely that the 3= ends of mK1 can be generated without anyprotein factors by nonconventional transcription termination.

mCONGRAD formation immediately after induction of thegal operon. So far, this study has demonstrated that a transcrip-tion termination event at the end of each cistron, galE, galT, galK,and galM, generates mE1, mT1, mK1, and mM1, as shown in Fig.1A. The stochastic nature in each of the transcription termina-tions suggests that a certain proportion of RNAP molecules thatleft the gal promoters would dissociate from DNA template at the

FIG 8 Specific location of the 3= ends of mK2. (A) The nested 3= RACE assaywas performed, and the five PCR primers (thicker arrows) and 21 extensionprimers (arrows) are shown. The nested 3= RACE assay was performed in 2different strains, the WT and rho mutant (rho::bla) strains. Numbers by eachband indicate the positions of the 3= ends relative to �1, the transcriptioninitiation site of the P1 promoter. (B) Only the 3= ends of mK2 are shown. (C)What is shown as mK1 3= ends are the results from 3= RACE assay performedon RNAs transcribed from the gal operon in vitro.

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end of each cistron. However, mK2 is produced by endoribonu-clease digestion of the pre-mK2. We questioned how fast and whattype of mCONGRAD would be formed as soon as the operonbecomes active in transcription. We tested this by measuring themCONGRAD after inducing derepression of the gal operon. Forthis, WT cells were grown in M9 minimal media with 0.5% glu-cose. When the growth reached an OD600 of 0.6, galactose wasadded to a final concentration of 0.5%.

Cells were taken at 1, 2, 3, and 4 min, and mCONGRAD wasmeasured. The results showed that after 1 min, only galE is in-duced (Fig. 9). After 2 min, the level of total gal transcripts was 4.5times that at time zero, suggesting that the operon can be active intranscription after addition of galactose even in the presence ofglucose. Nevertheless, after 2 min, type 2 mCONGRAD had al-ready been established. Assuming that the average speed of tran-scription by the sigma70 RNA polymerase is 42 nucleotides/s (22),it would take 1.8 min to transcribe the entire 4.2-kb gal operon.Thus, the formation of the type 2 mCONGRAD in 2 min after theinduction of transcription suggests that a single round of tran-scription is enough to establish mCONGRAD. These data are con-sistent with our hypothesis that stochastic transcription termina-tion is the primary cause of mCONGRAD formation. After 3 min,galK started to exceed galT, and type 1 mCONGRAD was estab-lished in 4 min (Fig. 9), suggesting that mK2 is being producedwhile mCONGRAD is formed. Since type 2 is established first afterone round of transcription and type 1 follows, it can be suggestedthat mK2 is produced posttranscriptionally.

However, it is not clear whether RNase P digestion occurs onlyon pre-mK2, whose transcription termination has occurred at the3= ends of mK2, or if any transcript that is cleaved by RNase P isterminated at the 3= ends of mK2.

DISCUSSION

The efficiency of transcription termination at each cistronjunction is less than 100%, and it is different from one cistronjunction to another. This is probably caused by a stochasticbehavior of individual RNA polymerase and/or transcriptionfactors on different DNA sequences involved in transcriptiontermination. It is the stochastic nature of transcription termi-nation that establishes the gradient in gene expression that ishighest in the first gene (thus, most proximal to the promoter),which is galE. Nature has maintained a gradient in gene expres-sion that is likely to have the first gene product (GalE) in the

largest amounts, and that is probably what the cell needs mostfor the catabolism of D-galactose in E. coli. One way for the E. colicells to change the gene expression of each cistron in the estab-lished natural polarity is to regulate the stochastic nature of tran-scription termination. These considerations raise the possibilitythat any factor(s) that could affect the stochastic nature of tran-scription termination would change gene expression establishedin natural polarity. It was suggested that Rho termination fre-quency can be modulated by altering elongation kinetics of RNAP(23). A very precise but subtle change in the expression of eachcistron (natural polarity) could be imagined if proteins or otherfactors regulate the elongation kinetics of RNAP according to theexternal or internal signals. In addition to the well-known proteinfactors, such as NusG and NusA, another group of proteins thatcan be oligomerized on DNA to form a scaffold, such as H-NS,Cnu, and Hha, was recently shown to participate in modulation ofRho termination efficiency (24).

The hexameric Rho protein requires an emerging mRNA re-gion known as a Rho utilization site (rut) for binding and functionas a transcription terminator. Typically, the rut site lacks certainsecondary structures and is composed of an �80 nucleotidestretch called the CG bubble that is rich in cytidine but poor inguanine residues (25). Using a computer algorithm (26), we iden-tified a CG bubble at each Rho-dependent termination site thatgenerates 3= ends of mE1, mT1, mK2, and mM1 (Fig. 10). Each CGbubble spans about 122, 90, 182, and 114 nucleotides, respectively,and appears upstream of the termination site. These data concurwith the current model of Rho termination that Rho binds tomRNA before acting upon RNAP to terminate transcription (27,28). An interesting observation is that there is an additional largerCG bubble, spanning 191 nucleotides, in the middle of the galEgene from �281 to �472 (Fig. 10, box). Since no transcriptiontermination activity after this CG bubble in the middle of galE hasbeen observed in WT cells and it is located in the middle of the firstgene of the operon, where 4 Rho terminations are expected tooccur downstream, we propose that this is where Rho is loaded

FIG 9 Kinetics of gal mRNA production after induction. MG1655 cells werecultured in M63 glucose medium to an OD600 of 0.6, and transcription fromthe gal operon was induced by addition of galactose to a final concentration of0.5%. Cells were harvested at 0, 1, 2, 3, and 4 min after induction. The gal-specific mRNA was measured using qRT-PCR, and the values relative to that ofthe galE at time zero are shown. The means standard deviations from at leastthree independent experiments are shown.

FIG 10 CG bubbles within the gal operon. Cytosine (black) and guanine(gray) frequencies of the nontemplate strand of the entire gal operon areshown below a map of the operon. The high-C, low-G region (CG bubble),known to be the binding site for Rho (rut), is also indicated. The CG bubblesthat lead to Rho-mediated termination are marked with ovals, and the first CGbubble at the start of the operon that does not lead to Rho-mediated termina-tion is marked with a rectangle. The upward-pointing arrows indicate thelocation of the 3= ends of gal-specific mRNAs. The Emboss Freak program(http://emboss.bioinformatics.nl/cgi-bin/emboss/freak), with a steppingvalue of 1 and an averaging window of 78, was used to calculate the frequenciesand outputs, which were exported to Microsoft Excel for graphical represen-tation.

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onto a paused RNAP for the terminations downstream. We actu-ally found a transcription pause in this CG bubble (data notshown). Thus, as transcription on the gal operon DNA progresses,RNAP that deploys the associated Rho through the CG bubbleidentified at the cistron junctions (circled in Fig. 10) would termi-nate transcription. These considerations raised the possibility thatthe stochastic nature of Rho termination came at the deploymentof Rho from RNAP.

Expression of galK can be controlled independently fromnatural polarity. The mCONGRAD status in the mid-log growthphase of the WT strain is type 1, where galK expression is greaterthan that of galT, forming a nonsmooth gradient of expression.The mCONGRAD in a cya strain is type 2, where galT expressionis greater than that of galK, forming a smooth gradient of expres-sion; thus, the order of expression is galE � galT � galK � galM,and there is almost no mK2 in the cya strain. These data suggestthat the difference between type 1 and type 2 mCONGRAD iscaused by mK2 production: more mK2 is produced in type 1 thanin type 2. As mentioned above, the stochastic nature of transcrip-tion termination at the end of each cistron establishes the type 2mCONGRAD. Thus, E. coli cells could regulate galK expressionover the already established mCONGRAD by regulating mK2production. Since it is the RNase P cleavage on a gal transcript thatinitiates mK2 production, one of the ways to produce more mK2so that cells can switch to type 1, where galK expression becomesgreater than that of galT, would be either to increase the synthesisrate or to decrease the decay rate of mK2. Note that since theRNase P digestion of a gal transcript would create a new 5= end infront of the galK gene and Spot 42 can bind to the 5= end of galKand decrease galK transcript levels (13, 14), it is likely that E. colicells regulate degradation of mK2.

Perhaps Spot 42 and Hfq, bound to a newly generated 5= end ofmK2, block translation and further engage in degradation of mK2by recruiting RNase E (29). The mK2 degradation by Spot 42 iscurrently being investigated. Since cAMP-CRP is a negative tran-scription factor for Spot 42 production (30), the data from thisstudy suggest that any external or internal changes that evokecAMP increase would not only promote the well-established no-tion of gal transcription initiation but also exert effects on expres-sion of galK only through mK2 production independently fromtranscription initiation and from natural polarity.

ACKNOWLEDGMENTS

This research was supported by the Basic Science Research Programthrough the National Research Foundation of Korea (NRF), funded bythe Ministry of Education, Science, and Technology (2013007271). Thisresearch was also supported by a grant from Chungnam National Univer-sity (2012-1689).

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