Antisense-RNA Mediated Transcriptional Attenuation: Importance … · 2012-11-30 · Importance of a U-turn Loop Structure in the Target RNA of Plasmid pIP501 for Efﬁcient Inhibition

Antisense-RNA Mediated Transcriptional Attenuation:Importance of a U-turn Loop Structure in the TargetRNA of Plasmid pIP501 for Efficient Inhibition by theAntisense RNA

Nadja Heidrich and Sabine Brantl*

Institut fur MolekularbiologieFriedrich-Schiller-UniversitatJena, Winzerlaer Straße, 10Jena D-07745, Germany

Antisense-RNA mediated gene regulation has been found and studied indetail mainly in prokaryotic accessory DNA elements. In spite of differentregulatory mechanisms, in all cases a rapid interaction between antisenseand target RNA has been shown to be crucial for efficient regulation.Recently, a sequence comparison revealed in 45 antisense RNA controlsystems a 50 YUNR motif indicative for the formation of a U-turn structurein either an antisense or a target RNA loop and confirmed in the case ofthe hok/sok system of plasmid R1 its importance for regulation.

Here, we demonstrate the importance of the 50 YUNR motif in the targetRNA (RNAII) loop L1 of the replication control system of plasmid pIP501.The effect of four individual mutations in L1 was studied in vivo and invitro. Mutations that maintained the putative U-turn or swapped it fromsense to antisense RNA were silent, whereas mutations that eliminatedthe 50-YUNR motif showed two- to threefold elevated copy numbers invivo in correlation with three- to fourfold reduced inhibition rate constantsof the complementary RNAIII species in vitro, whereas the half-lives of allRNAIII species were not affected. ENU probing experiments confirmedthe U-turn structure for the silent mutation (N-C) and disruption of thisstructure upon alteration of the invariant U or inversion of the YUNRmotif-containing loop. RNA secondary structure probing excluded loopsize alterations as a reason for altered inhibition rates. Implications forthe pathway and efficiency of RNAII/RNAIII interaction, and hence,pIP501 copy-number control, are discussed.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: antisense-RNA mediated transcriptional attenuation; sense/antisense-RNA interaction; U-turn loop structure; plasmid copy-numbercontrol; Gram positive bacteria*Corresponding author

Introduction

Antisense-RNA mediated gene regulation hasbeen found and studied mainly in prokaryoticaccessory DNA elements like plasmids, phagesand transposons.1,2 In all these cases, the antisenseRNAs exert their inhibitory function by a varietyof different mechanisms, among them inhibitionof translation, prevention of primer maturation,transcriptional attenuation and inhibition of for-mation of an activator pseudoknot. Over the recent

few years, a few chromosomally encoded antisenseRNAs have been identified and characterized,3 – 6

and, very recently, three groups found in total 45novel small non-coding RNAs with hithertounknown function.7 – 9 At least some of them will,certainly, turn out to be antisense RNAs, and thefunction of one of these novel RNAs has beenrecently elucidated.10

In many cases, the kinetics of antisense RNA/sense RNA interaction were analysed and bindingpathways were proposed. Binding initiates mainlyby loop-loop contacts (plasmid copy number con-trol systems) or linear region-loop contacts (hok/sok, IS10).1,11 Independent of the individual inhibi-tory mechanism or the binding pathway, efficient

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviation used: ENU, ethylnitrosourea.

doi:10.1016/j.jmb.2003.09.020 J. Mol. Biol. (2003) 333, 917–929

inhibition relies on a rapid bi-molecular interactionbetween the sense and the antisense RNA. Pairingrate-constants were calculated and found to bemainly in the range of 106 M21 s21.1 Structuralrequirements for efficient antisense RNAs havebeen determined:12,13 loops must be GC-rich andcomprise 5–8 nt, whereas stems longer than 10 bpshould contain bulges to prevent RNase III diges-tion and to allow efficient pairing.

Recently, a comparative sequence analysisrevealed in 45 well-studied cases either in a senseor antisense RNA loop the sequence motif 50-YUNR in position 1–4 of 6 nt and 7 nt loops andin position 2–5 of 8 nt and 9 nt loops.14 This motifis predicted to form a U-turn, an RNA structuralmotif that was first identified in the anticodonloop and the TcC-loop of yeast PhetRNA,15 and haslater also been found in the crystal structure of thehammerhead ribozyme16 and in the GNRAtetraloop.17 Furthermore, it was found in severalrRNAs, e.g. very recently, in the 23 S rRNA hairpin35,18 in IIa loop of yeast U2 RNA19 and the HIV1LystRNA acceptor loop.20 In all these cases, NMRor crystal structure analyses revealed a sharp bendin the sugar–phosphate backbone that presentsthe following three or four bases in a solventexposed, stacked configuration, a half A-formWatson–Crick structure, providing a scaffold forrapid interaction with complementary RNA. Thechange in backbone direction is stabilized by oneor two contacts across the bend: one hydrogenbond between N3 of uracil and a phosphate back-bone oxygen atom 30 of the UNR sequence, andthe other between the 20 OH-group of uracil andN7 of the YUNR purine. The majority of the experi-mentally determined U-turns have been associatedwith four tertiary contacts,21 but, with the work ofFranch et al. they are now inferred in the formationof RNA/RNA interactions in natural antisenseRNAs. Franch et al. analysed the significance ofthe putative U-turn in the sense-RNA loop of thehok-mRNA of plasmid R1. Their results show thatelimination of the 50-YUNR motif resulted in sig-nificantly reduced antisense-RNA pairing kinetics,whereas mutations maintaining the motif weresilent. ENU probing experiments confirmed theU-turn structure for the wild-type and showed analtered loop structure in the case of non-silentmutations. Based on these results and sequencecomparison data, the authors proposed that theU-turn motif might be a generally employedenhancer of RNA pairing rates.

Plasmid pIP501 belongs, together with pAMb1and pSM19035, to the inc18 family of theta replicat-ing broad host range streptococcal plasmids.22

Replication control is exerted by two inhibitorycomponents, an antisense RNA (RNAIII) and atranscriptional repressor (CopR).23,24 RNAIII actsby transcriptional attenuation of the essentialrepR-mRNA,25 a control mechanism so far onlyfound in Gram positive bacteria. Previously, wemapped the secondary structures of RNAIII andRNAII and determined the pairing rate constants

of the RNAII/RNAIII pair as well as the inhibitionrate constants of RNAIII.26 Inhibition occurs tentimes faster than stable pairing, indicating thatsteps preceding formation of a full duplex are suf-ficient for inhibition.26 Furthermore, the analysis offour mutants in different regions of RNAIIIsuggested that loop L3 of RNAIII might be the rec-ognition loop that makes the first contact with thetarget RNA, since mutations in this loop led tonew incompatibility groups.27

Inspired by the comparative sequence analysis ofFranch et al. who reported a 50 YUNR motif (50

CUGA) in the 50 loop (L1) of the target RNA(RNAII) of pIP501, and by the results obtained bythe authors for the hok/sok system, we set out toanalyse the significance of this potential U-turnforming motif for the interaction with the antisenseRNA, RNAIII, and hence, the regulation of pIP501replication. Here, we report a detailed in vivo andin vitro analysis of wild-type RNAII/RNAIII andfour 50-YUNR mutants, two of them designed toprevent formation of the U-turn, one silentmutation and a flip-flop mutation transferring the50 YUNR motif from sense to antisense RNA. Ourresults indicate that this motif is important for thestructure of the 50 loop of RNAII and for the effi-ciency of transcriptional attenuation, and, there-fore, copy number control.

Results

Design of mutants in the 50 YUNR motif

We decided to construct the following fourmutants which contain alterations in the 50 term-inal loop L1 of the sense RNA, RNAII. In mutantpPRC1/1, the invariant U in position 34922

(see also numbering in Figure 1A), was replacedby A, which should result in an altered loopstructure. In mutant pPRC3/1 (loop inversionmutant), the entire loop containing the 50 YUNRmotif was inverted resulting in 30 YUNR (nucleo-tide position 347–355 CCUGAGAAA replaced byAAAGAGUCC). As negative controls that are pre-dicted to behave like the wild-type, a silentmutation was designed in which the N of YUNRwas altered from G (350) to C (pPRC4/1) and aflip-flop mutant, where the corresponding loops ofthe antisense and the sense RNA were exchanged(pPRC5/1, nucleotide position 347–355 CCUGA-GAAA replaced by UUCUCAGG). Here, pPRC/1,which was constructed in the same way as allmutated plasmids analysed here, was used as thewild-type in all assays. Figure 1C shows a sche-matic representation of the RNAII L1 loops ofwild-type and all constructed mutants (Table 1).

Activity of the RNAII/RNAIII mutants in Bacillussubtilis in copy number control

All mutants were analysed for their in vivo effecton the regulation of replication of the corresponding

918 Importance of a U-turn Loop Structure in pIP501 Target RNA

pIP501 derivatives. B. subtilis strain DB104 wastransformed with the corresponding pIP501derivatives and copy numbers of steady state-cul-tures were determined as described in Materialsand Methods. The corresponding gels are shownin Figure 2, and the results are summarized in

Table 2. Both pPRC1/1 and pPRC3/1 containingthe U-A and the loop inversion mutations, repli-cated at 2.5 to threefold higher copy numbers thanthe wild-type. In contrast, pPRC4/1 and pPRC5/1containing the silent and the flip-flop-mutation,respectively, replicated at wild-type like copy num-bers. Apparently, the introduction of the U-A and30 YUNR mutations in the 50 YUNR motif led to amoderate, but significant effect on replication, indi-cating that this motif is of importance for copy-number control. On the other hand, the resultsobtained with the silent and the flip-flop mutationwere as expected, i.e. replication control was notaffected when the 50 YUNR motif was conservedor was present on the complementary antisense-RNA loop instead of the sense-RNA loop.

Half-lives of mutant RNAIII species in Bacillussubtilis

To rule out that half-life alterations caused by theintroduced mutations were, in part, responsible forthe observed copy number effects, all mutatedrnaIII genes were subcloned into vector pGK14 toallow the determination of RNAIII half-lives in the

Figure 1. Schematic represen-tation of the RNAII/RNAIII inter-action. A, Complete sequence ofRNAIII. The two relevant loops L3and L4 are underlined. Each tenthnt is shown in bold to facilitatenumbering. B, Schematic drawingof the interacting RNAII andRNAIII. Loops L1-L4 of RNAIIIand loops L1 and L2 of RNAII areindicated. Black circles specify thelocation of the 50-YUNR motif. C,Computer predictions of 50-loops(L1) of the sense RNA (RNAII)species are shown. Loop sizes andstructures were confirmed later bystructure probing and ENU foot-printing experiments (see Figures 3and 4).

Figure 2. Copy number determination of wild-typeand mutant pIP501 derivatives in B. subtilis. Separationon 1% agarose gels of Bam HI linearized aliquots ofundiluted plasmid DNAs prepared from 1 ml culturevolumes of B. subtilis strains containing wild-type ormutant pIP501 derivatives grown to the same opticaldensity in late logarithmic phase. In all cases, five trans-formants grown in parallel were used for plasmid prep-arations. Gel photos were scanned and quantified usingthe PCBAS 1.0 software. The results are shown in Table 3.

Importance of a U-turn Loop Structure in pIP501 Target RNA 919

absence of RNAII. The resulting mutant plasmidspGKUA, pGKLI, pGKFF, pGKNC along with pre-viously constructed wild-type derivative pGKIII/1were introduced into B. subtilis DB104, transfor-mants grown in minimal medium and half-lives ofall RNA species determined as described before.27

The results (data not shown) confirmed that allhalf-lives were with five minutes to seven minutesin the same range. Therefore, the altered copynumbers could not be attributed to altered anti-sense-RNA half-lives.

Structures of wild-type and mutant sense RNAloops as revealed by ethylnitrosourea(ENU) probing

To confirm the U-turn structure and to find outwhether the mutations altering the loop sequenceor direction of the proposed U-turn motif causedstructural changes, we tested wild-type and mutantloops for the accessibility of the phosphate backboneto alkylation by ethylnitrosourea (ENU) (Figure 3;

Table 3). ENU was chosen since most other chemicalprobes are sensitive to sequence differences.

In both the wild-type and the N-C (silentmutation) loop L1 structure, backbone alkylationwas reduced at the ApGpA phosphate groups 30

of the U residue. Additionally, in the case of thesilent mutation, strong alkylation was observed atUpC (arrow in Figure 3 and Table 3), supporting asolvent-exposed turning phosphate group. This isin good agreement with the formation of a U-turnstructure for the loop L1 of the N-C mutant. In con-trast, the U-A mutant loop showed a reduction ofalkylation at the turning phosphate ApG, followedby a slight increase in backbone alkylation at thepositions 30 of ApG. The loop inversion mutantrevealed no significant alteration in phosphatebackbone alkylation throughout the entire loop.Since the flip-flop mutant sense-RNA loop L1 is infact the wild-type antisense-RNA loop L3, noalterations in the backbone alkylation pattern wereexpected. As shown in Figure 3 and Table 3, aslight reduction at the position of the turning phos-phate, followed by an increase in the alkylation atthe two 30 positions, experimentally verified thisassumption. These data indicate that the U-A andloop inversion mutations indeed caused alterationsin the structure of the RNAII loop L1 containingthe 50 YUNR motif. In contrast, the silent mutationthat behaved like the wild-type in all assays (seealso below), clearly showed a backbone alkylationpattern supporting formation of a U-turn structure.In the wild-type case, we cannot decide from theENU data, whether or not a U turn is formed.

Table 1. Plasmids used in this study

Plasmid Description Reference

pUC19 E. coli cloning vector, ApR, MCS 40pPR1 Shuttle vector, pIP501 derivative lacking the cop R

sequence upstream of the Kpn I site, ApR, PmR

23

pPR4 pPR1 derivative lacking promoter pII 23pCOP1B2 pPR1 derivative carrying the copR gene downstream from

oriR, PmR

24

pGK14 Broad host range lactococcal vector, EmR 24pGKIII/1 pGK14 containing the wild-type rnaIII gene as EcoR I

fragment, EmR

27

pUCU1 pUC19 derivative comprising nt 160–582 of the pIP501replicon as Bam HI/Pst I fragment

This study

pUCR1 pUC19 derivative comprising nt 582–1632 of the pIP501replicon as Pst I/Hind III fragment

This study

pUCR3 pUC19 derivative comprising the pUCR1 Pst I/Hindfragment and the pPR1 Eco RI/Hind III fragment

This study

pPRP pPR4 derivative carrying the pUCU1 Bam HI/Pst Ifragment and the pUCR3 Pst I/Eco RI fragment

This study

pPRC/1 pPRP with the 549 bp copR fragment from pCOP1B2 This studypPRC1/1 pPRC derivative with the U349A mutationa This studypPRC3/1 pPRC derivative with inversion of nt 347–352a This studypPRC4/1 pPRC derivative with G350C mutationa This studypPRC5/1 pPRC derivative with complementary sequence of

RNAIII between nt 347 and nt 356This study

pGKUAa As pGKIII/1, but rnaIII gene from pPRC1/1 This studypGKLI As pGKIII/1, but rnaIII gene from pPRC3/1 This studypGKNCa As pGKIII/1, but rnaIII gene from pPRC4/1 This studypGKFf As pGKIII/1, but rnaIII gene from pPRC5/1 This study

a Mutations as manifested in the RNAII sequence.

Table 2. Comparison of copy numbers of wild-type andmutant pIP501 derivatives in B. subtilis

Plasmid Relative copy number

pPRC/1 £pPRC1/1 2.5 £pPRC3/1 2.5–3 £pPRC4/1 £pPRC5/1 £


Efficiency of transcription termination in vitromediated by wild-type and mutantRNAIII species

As shown previously for plasmid pIP501, inhi-bition by the antisense RNA (transcriptional ter-mination) occurs about ten times faster thanformation of stable duplexes between antisenseand sense-RNA,26 indicating that steps precedingstable pairing are sufficient for inhibition. Therefore,we used our previously developed single-roundtranscription assay26,27 that allows to determineinhibition rate constants to analyse the efficiencyof mutated RNAIII species to interact with theircomplementary targets. This assay will detectdifferences that are not found in a simple duplexformation analysis. It has the following properties:first, sense-RNA transcription starts simul-taneously, and restarts are prevented, so that thefate of the nascent RNA can be followed in time.Second, antisense-RNA is not transcribed in this

assay, so that the effect of defined concentrationsof added in vitro synthesized [3H]UTP labelledRNAIII on the termination frequency can beassessed. Third, for the calculation of a rate con-stant of inhibition, the time window during whichthe antisense-RNA can exert its effect, had beendetermined before to be <15 seconds.26 500 bpDNA fragments spanning the pII/pIII region, theattenuator and 100 bp downstream were generatedby PCR using pPRC/1, pPRC1/1, pPRC3/1,pPRC4/1 and pPRC5/1 as templates in the single-round in vitro transcription with B. subtilis RNApolymerase. In a preincubation step, repR-RNAtranscription was primed by inclusion of the dinu-cleotide ApA, [a-32P]ATP, low concentrations ofATP and CTP and RNA polymerase (see Materialsand Methods). This confined initiation complexformation to the repR promoter pII, because ApAis unable to function as a start dinucleotide atthe antisense-RNA promoter pIII.28 The omissionof two NTPs (UTP, GTP) stalls the initiation

Figure 3. Ethylnitrosourea (ENU) probing analysis of wild-type and mutant RNAII loop structures. Probing wasdone on 50-end 32P-labelled RNAII species carrying the loop sequences shown in Figure 1. T1, RNase T1 cleavage reac-tion (T1 cleaves 30 of unpaired G residues); T2, RNase T2 cleavage reaction (T2 cleaves unpaired nucleotides with aslight preference for A residues); L, alkaline ladder. The wild-type loop sequence is shown for backbone cleavage local-ization. Above each lane the presence (þ) or absence (2) of ENU and the assay temperatures are indicated. The levelof alkylation was evaluated by comparing alkylation (37 8C) to that of the corresponding denatured RNA (90 8C) nor-malized with the ratio of alklylation of the denatured and renatured RNAs in the stem regions of the RNA hairpin.Note that the alkylated RNAs exhibit a slightly reduced mobility as compared to that of the unmodified RNA in theT1/T2 cleavage reactions and the alkaline ladder (L).

Table 3. Quantification of band intensities of wild-type and mutant RNAII loops as revealed by ENU probing

nt nt-Position RNAIIWT RNAIIU-A RNAIILi RNAIINC RNAIIFF

A 355 0.860 0.620 0.854 0.759 0.893A 354 0.650 0.640 0.945 0.731 0.869A 353 0.593p 0.900 0.972 0.764p 0.890G 352 0.847p 0.920 1.000 0.889p 1.035A 351 0.954p 0.954 1.040 0.985p 1.105G 350 1.090 0.860 1.04 1.489 " 0.930U 349 1.290 0.940 0.963 1.053 1.010C 348 1.306 2.200 1.010 0.988 0.987C 347 1.190 0.920 1.050 1.072 1.026

The alkylation pattern of all single stranded nucleotides of the 9 nt loop L1 of RNAII is shown. Nucleotides forming the 50 YUNRmotif are shown in bold, the invariant U is underlined. The level of alkylation was evaluated by comparing alkylation (37 8C) to thatof the corresponding denatured RNA (90 8C) normalized with the ratio of alkylation of the denatured and renatured RNAs in thestem regions of the RNA hairpin. An asterisk corresponds to $25% reduction in alkylation, whereas an arrow indicates an increasein alkylation.


complexes after incorporation of only a fewnucleotides. After five minutes incubation at30 8C, aliquots of the preincubation mix wereadded to prewarmed tubes containing wild-typeor mutated RNAIII species in the range from5 £ 1029 M to 3 £ 1027 M, and elongation of RNAIItranscription was started by addition of solution 2containing all four unlabelled NTPs and rifampicinto limit the analysis to synchronous single-rounds.Transcription was assayed at a ten minute timepoint by separating the samples on denaturingPAA gels. As shown in Figure 4, increasing concen-trations of antisense RNA induced progressivelyincreased termination of the target RNA (T versusF bands, Figure 4). As control, three parallel incu-bations were performed in the absence of RNAIII(buffer controls). Figure 4A–E shows the Phos-phorImager outprints of corresponding gels forthe wild-type and all mutants. In all cases, a

quantitation of the assay is presented below,which was used to calculate the inhibition rate con-stants of all RNAIII species as described below.

Calculation of the rate constants of inhibition

The rate constants of inhibition were calculatedas described previously26 using the followingequation:

kinhib ¼ln 100 2 lnðNð½t�Þ

½RNAIII�t

where t is the time window for inhibition (here:15 seconds), NðtÞ is the percentage of repR-RNAtranscripts that escaped inhibition at time t (Fband ¼ “full-length” transcript) determined fromgels as in Figure 4A–E, and [RNAIII] is the con-centration of the antisense RNA (RNAIII species)

Figure 4. The effect of different concentrations of wild-type and mutant RNAIII on attenuation in vitro. In vitroattenuation assays were performed with wild-type (A), U-A mutant (B), loop inversion mutant (C), N-C-mutant (D),and flip-flop mutant (E). RNAIII species were included at the concentrations indicated (bottom panel) and their effectson induced transcription termination determined at a ten minute time point. The upper part shows PhosphorImageroutprints with the positions of full-length (run-off repR-RNA, <365 nt; F) and terminated repR-RNA (<260 nt, T).The lower part shows staple diagrams based on PhosphorImager quantifications of the signals derived from theupper part. Calculations and background substractions were as described in Materials and Methods. Three buffer con-trols give the value in the absence of antisense RNA. The average intensity of the F band in the buffer controls (as F/total F þ T]) was set to 100% (regulatable F). Values given for the incubations containing RNAIII represent: percentageof F band intensity measured/F band intensity of the buffer control, corrected for the sum of total T and F band inten-sity. Antisense-RNA concentrations are indicated. M, pBR322 MspI size marker. Arrows indicate the increase in theconcentration of RNAIII.


present. At the highest and lowest concentrationsof RNAIII used, large variations in the calculatedinhibition rate constants were observed. In thefirst case, a minor fraction of termination incompe-tent molecules may obscure the measurements,and in the latter case, intrinsic RNAIII independenttermination (see buffer controls) varies slightly,and therefore, a small inihibition at low RNAIIIconcentrations cannot be measured exactly. There-fore, all calculations were based on experimentswhere the degree of inhibition was between 20%and 80%, and RNAIII was present at 1 £ 1028 M to6 £ 1028 M. Table 4 summarizes the results. Itshows that the inhibition rate constants ofRNAIIIU-A and RNAIIILi carrying an altered U-turnstructure, were three- to fourfold lower than thatof the wild-type. In contrast, RNAIIIN-C andRNAIIIFf exhibited only slightly lower (60% or72% of the wild-type values) inhibition rate con-stants. These data are in good correlation with theabsence and presence of the U-turn structureshown by ENU footprinting and with the deter-mined copy numbers.

Since altered inhibition rates might not only becaused by altered loop structures, but also byaltered loop sizes,12 secondary structure probingexperiments had to be performed to prove that theintroduction of the 50 YUNR-mutations did notalter the sizes of the interacting RNAII/RNAIIIloops.

Secondary structures of the interacting loopsof mutant and wild-type RNAIII andRNAII species

Secondary structure probing experiments of therelevant interacting loops L2 (RNAII) and L4(RNAIII) were performed as follows: RNAII andRNAIII species were synthesized in vitro by T7RNA polymerase, 50-end-labelled, gel purified andsubjected to partial digestions with RNases T1(cleaves 30 of unpaired G residues), T2 (unpairednucleotides with a slight preference for A residues)and V1 (double-stranded and stacked regions).Figure 5A summarizes the results. Schematic rep-resentations of structures consistent with the clea-vage data are presented in Figure 5B.

Loops L1 of RNAIIWT, RNAIIU-A and RNAIILi

showed strong T1 cuts 30 of G350 and G352,

whereas only in the wild-type and the loop inver-sion case a weak cut could be detected for G346.T2 cuts between 347 and 355 indicated a loop sizeof nine nucleotides for RNAIIWT and RNAIIN-C.The latter one showed, due to the altered loopsequence (G350C), only one strong T1 cut 30 ofG352. Although the 50 loops of RNAIIU-A andRNAIILi exhibited somewhat fewer T2 cuts thanthose of wild-type and N-C mutant, the combi-nation of T2 and V1 cleavage results supports 9 ntloops in these cases as well. RNAIIFf contains theantisense RNAIII loop L4 instead of RNAII loopL1, and hence, has an altered loop sequence andsize: no T1 cuts were observed as expected, andsix T2 cuts together with a series of V1 cuts on the50 half of the stem indicated the presence of a 7 ntloop. From these data we can conclude thatRNAII-loop sizes were not altered upon introduc-tion of the U-A, N-C or loop inversion mutation.

For loop L4 of the RNAIII species, the followingsecondary structure probing results were obtained.RNAIIIWT showed two strong (nt 352 and 353) andfour weak T2 cuts (nt 349, 351 and 354) andRNAIIIU-A five strong (nt 350–354) and one weakT2 cuts (nt 349) together with a strong T1 cut 30 ofnt 348 supporting a loop size of seven nucleotidesfor the wild-type and the U-A mutant. In RNAIIIU-A,a weak T2 cut 30 of nt 355 together with a V1 cutat this position indicated a certain breathing at thebasis of this loop. In the case of RNAIIILi, fourstrong and two weak T2 cuts also supported a 7 ntloop L4. In these three RNAIII species, clear V1cuts at positions 356–360 (U-A and Li) and 356–359 (WT) 50 of L4 confirmed the 7 nt loop sizes. Inthe case of RNAIIIFf, a 9 nt loop (identical withthat of RNAII loop L1) was supported by threestrong and four weak T2 cuts and two strong T1cuts. RNAIIIN-C revealed the same structure andloop size as wild-type and U-A mutant (data notshown). Consequently, the loop size of RNAIIIloop L4 was not changed by the introduction ofthe U-A, N-C or loop inversion mutation.

In summary, we can conclude that the intro-duced mutations did not alter the sizes of the rele-vant interacting loops, i.e. in all cases, a 9 nt loopinteracts with a 7 nt loop. Thereby, in the wild-type, U-A, loop inversion and N-C cases thesense-RNAs carry the 9 nt loops and the anti-sense-RNAs the 7 nt loops, and in the flip-flopmutant it is vice versa.

Discussion

Here, we present a detailed in vivo and in vitroanalysis on the role of the 50 YUNR motif (50

CUGA) found in position 2–5 of loop L1 of thesense RNA (RNAII) of plasmid pIP501. This ana-lysis was aimed at the elucidation of the import-ance of this motif for the efficiency of RNAII/RNAIII interaction, and hence, regulation ofpIP501 replication. Four RNAII loop mutants weredesigned. Two mutations should alter the putative

Table 4. Inhibition rate constants of wild-type andmutant RNAIII species in vitro

RNAIIIspecies

Inhibition rate constantkinhib (M21 s21)

Relativekinhib

RNAIIIWT 3.3 £ 106 1 £RNAIIIU-A 0.9 £ 106 0.27 £RNAIIILi 0.73 £ 106 0.22 £RNAIIIN-C 2.4 £ 106 0.72 £RNAIIIFf 2.0 £ 106 0.60 £

All indicated values are the average results of four parallel,independently performed experiments.


U-turn structure (U349A and loop inversionleading to 30 YUNR), one mutation was proposedto be silent (G350C ¼ N-C) and in the fourth loopswapping between RNAII and RNAIII transferredthe 50-YUNR motif to RNAIII loop L4 (flip-flop).Copy number determinations revealed that thismotif seems to be important for the regulation ofplasmid replication. Although the observed effectsof the U-A and the loop inversion mutation wereonly moderate (two- to threefold copy numberincreases), they were significant in that the twocontrols (N-C and flip-flop) behaved as the wild-type (Figure 2; Table 2). Half-life determinations ofall RNA species (data not shown) confirmed thatthe introduced mutations did not affect the RNAIIIhalf-life. Therefore, only alterations of the structureor size of the corresponding loops could be respon-sible for the observed copy number effects. To dis-tinguish between these two possibilities, ENUfootprinting was used to investigate structural

alterations introduced by the mutations, whereasRNA secondary structure probing was used tomap loop sizes. The results of the ENU probing(Figure 3; Table 3) for the silent mutation (N-C)were indeed consistent with formation of a U-turnstructure: high accessibility of the UpC backbonesupporting a solvent-exposed turning phosphategroup and low accessibility of the ApGpA phos-phate groups 30 of the U residue. In the wild-type,low accessibility of the ApGpA phosphate groupswas not accompanied by high accessibility of theUpG backbone. Therefore, we cannot unequivo-cally decide from the ENU data whether or not aU-turn is formed in the wild-type case. However,the two mutants U-A and Li (loop inversion)clearly showed an altered loop architecture in com-parison with both wild-type and N-C mutant(Figure 3 and Table 3). It is necessary to mentionthat ENU footprinting did not in all cases confirmexisting U-turns, i.e. in tRNA, where the U-turn of

Figure 5. Secondary structures of RNAIII and RNAII species. A, Secondary structure probing of the 30-loops L3 andL4 of wild-type and mutant RNAIII species and of 50 loops L1 of wild-type and mutant RNAII species with threedifferent RNases. Purified, 50-end-labelled RNA species were subjected to limited cleavage with RNases as indicatedand separated on 8% denaturing gels. PhosphorImager outprints are shown. RNase concentrations used were: RNaseT1, 1021 and 1022 U/ml (in RNAIIIWT additionally 1023 U/ml); RNase T2, 1022 U/ml and 1023 U/ml (in RNAIIIWT

additionally 0.3 £ 1023 U/ml), RNase V, 1021 U/ml, 1022 U/ml and 2 £ 1022 U/ml. L, alkaline ladder, C, control with-out RNases, M, pBR322 MspI marker. No RNase V analyses were performed for the RNAIIU-A. Therefore, no V-signalcould be included into the stem of the stem loop in B. B, Proposed secondary structures of RNAIIIWT, RNAIIIU-A,RNAIIILi and RNAIIIFf (left) and RNAIIWT, RNAIILi, RNAIIU-A, RNAIIFf and RNAIIN-C (right) based on the cleavagedata and on additional experiments (data not shown) are depicted. Major and minor cuts are indicated by symbols(see box).


the anticodon loop was known from the crystalstructure, it could not be found by ENU probing(P. Romby, personal communication). Similarly, inthe target loop CopT of plasmid R1, all experimen-tal data unequivocally supported the existence ofa U-turn, except ENU probing.29 Therefore, thefinal proof for the existence of a U-turn structurecan be only a crystal or an NMR structure of thewild-type 50 loop L1 of pIP501 RNAII.

Secondary structure probing of the relevantsense and antisense RNA loops revealed that noloop size alterations were caused by the U-A, N-Cand loop inversion mutations. In the flip-flopmutation, the flipped loops had the expectedsizes, too (Figure 5). Consequently, in all cases, a7 nt loop should interact with a 9 nt loop, wherebythe 9 nt loop contains the wild-type or mutated50-YUNR motif at nt positions 2–5.

Half-life determination, ENU probing and RNAsecondary structure probing unequivocallydemonstrated that the introduced mutations chan-ged exclusively the structure of the 50-loop L1 ofRNAII. Observed copy number effects are, there-fore, due to altered RNAII loop L1 structures.Since copy number regulation in pIP501 occurs byantisense-RNA driven transcriptional attenuation,we asked, whether the altered loop structuresaffected the transcription termination efficiencies.A previously developed in vitro attenuation assaywas used to analyse transcription terminationcaused by wild-type or mutated RNAIII speciesand to calculate RNAIII inhibition rate constants.The results showed a clear correlation betweeninhibition rate constants and copy numbers. In thecase of the U-A-mutant, a threefold decreased inhi-bition rate was accompanied by twofold increasedcopy number, and for the loop inversion mutant,a fourfold decreased inhibition rate wasaccompanied by a threefold increased copy num-ber. On the other hand, the silent mutation thatreplicated at wild-type copy number had only aslightly reduced inhibition rate-constant comparedto the wild-type. Furthermore, the flip-flop mutantthat carried the U-turn motif on the antisenseinstead of the sense loop and replicated at wild-type copy number, was also almost as efficient ininhibition as the wild-type. This indicates that oneU-turn containing loop-independent, whethersense or antisense, RNA loop, is required for arapid bi-molecular interaction between sense andantisense RNA to yield efficient transcriptionalattenuation of pIP501 RNAII. The inhibition rateconstant determined in this study for the wild-type RNAIII was slightly higher than that deter-mined previously (3.3 £ 106 M21 s21 versus1–2 £ 106 M21 s21). This is most likely due to theuse of a new preparation of B. subtilis RNA poly-merase and a slightly altered transcription buffer(120 mM Tris–HCl, 25 mM MgCl2, 2 mM DTT; seeMaterials and Methods).

We cannot, at this stage, decide whether RNAIII-L4/RNAII-L1 contacts are of the same importanceas RNAIII-L3/RNAII-L2 contacts.

Formerly, we proposed that the primary inter-action between RNAII and RNAIII starts at the 30

loop L2 of RNAII and loop L3 of RNAIII27 (Figure1A). This hypothesis was not only based onmutations in these two complementary loops,which led to increased copy numbers and newincompatibility groups and altered the pairingbehaviour of the corresponding mutant RNAIIIspecies (382 and 383) towards wild-type RNAII,but also on RNAIII-L4 mutant 347. As we knownow from our detailed secondary structure prob-ing experiments, the latter mutation used at thattime to analyse effects on loop L1 of RNAII (theone with the U-turn-motif) and L4 of RNAIIIaffected the loop closing base-pair (G347A ofRNAIII) without altering loop size or sequence.This was, most likely, the reason why mutant 347behaved as the wild-type in the incompatibilitytests and duplex formation analyses, whereas itscopy number effect was, probably, the result of asecond mutation in the stem of loop L4 (insertionof an U between 344 and 345). The data obtainedin the course of this study suggest that interactionsbetween RNAII-L1 and RNAIII-L4 are at least asimportant as those between RNAII-L2 andRNAIII-L3. Although we do not yet have anyknowledge on the binding pathway betweenRNAII and RNAIII, we hypothesize that a simul-taneous interaction between two complementaryloop pairs is required for formation of the firstbinding intermediate (kissing complex). Alterna-tively, binding could start at RNAII-L1/RNAIII-L4and proceed in one direction (directed by theU-turn in RNAII-L1), and, later, involve the inter-action between RNAII-L2 and RNAIII-L3. For-merly, we discussed a third alternative: RNAIIIstem-loops L3 and L4 may be necessary to keepthe unstructured region in an accessible confor-mation to permit base-pairing within this regionto initiate and subsequently propagate tocompletion.26 However, duplex formation analyseswith wild-type RNAII L1 and RNAIII loop L3mutants performed before27 and L4 mutants per-formed in the course of this study (data notshown), indicate that the first critical steps involvenucleotides within the loops RNAIII-L3/RNAII-L2and RNAIII-L4/RNAII-L1 (the latter one contain-ing the YUNR motif) and that, most probably, thesingle-stranded region serves as a nucleation siteto allow persistent duplex formation. This wouldbe in good agreement with data obtained for pair-ing between CopA and CopT of plasmid R1. Here,the initial interaction occurs between two loops,1

whereby the target loop contains the U-turn motif.Recently, the binding pathway between CopA andCopT has been elucidated in detail: Binding startswith the interaction of two single loops of CopAand CopT. Later, a single stranded region isrequired to overcome the torsional stress createdupon progressing of this loop-loop interaction.Next, a partial duplex is formed which contains afour-helical junction.30,31 This intermediate is con-verted into the stable inhibitory complex which is


only a partial duplex and is only slowly convertedinto a stable duplex, the substrate for RNase IIIdegradation (see Wagner et al.1 for Figure ).Recently, the importance of the U-turn motif in themajor loop of CopT 50 UUGG (nt 1–4 of 6 nt loop)for efficient sense/antisense-RNA interaction and,hence, regulation, has gained experimental evi-dence. When the orientation of the 50-YUNRsequence was inverted, CopA/CopT binding ratesin vitro were decreased more than tenfold, paral-leled by a corresponding regulation defect in vivo.In contrast, loop swapping between CopA andCopT (flip-flop mutation) maintained wild-type-like behaviour.29 In the R1 hok/sok-system, where,in contrast to CopA/CopT and RNAII/III ofpIP501, a loop/tail interaction is the first criticalinteraction, U-A, U-G and U-C substitutions in theU-turn motif of the hok-mRNA loop that disruptedthe U-turn structure led to tenfold, 15-fold or three-fold impaired pairing rates with Sok-antisense-RNA,14 respectively, whereas mutations thatretained the U-turn, remained silent. Duplex for-mation as assayed in this study reflected the rateof initial pairing between hok-RNA loop and Sok-RNA tail, i.e. the committed step. In the case ofColE1, some RNAI loop inversions analysed byTomizawa’s group more than ten years ago alsodisrupted the 50 YUNR motif and led to a 25-foldreduction in binding rates.32,33 The same wasobserved for an increase in interstrand GC content,which disrupted the YUNR motif in IncI/repZ ofColIB-P9.34,35 In the case of RNA-OUT, substitutionof the invariant U by A in the YUNR motif yieldeda fivefold reduction in the pairing rate with RNA-IN from IS10.36 Although formation of a U-turnstructure was experimentally supported only inthe case of hok/sok, in all these systems, R1CopA/CopT and hok/sok, ColE1, ColIB-P9 andIS10-mutations in the YUNR motif led to signifi-cant (five to 30-fold) effects on RNA/RNA inter-action, suggesting that the U-turn structure is animportant denominator of rapid bi-molecularinteractions.

A recent analysis on structural features of F plas-mid FinP/traJ interactions which, as the authorspropose, also starts with loop-loop contactsshowed that mutations in stem loop SL-I of FinPthat altered the YUNR motif (50 CUCA to 50

GACA or 50 CAGU) led to only twofold decreasesin pairing rates,37 which were overcome by the FinOprotein. However, in both mutants, one additionalnucleotide adjacent to the YUNR motif was altered,too, which could have interfered with the effects ofthe pure U-turn mutations (compensation for alteredloop size or structure). Therefore, it cannot be saidunequivocally that the sequence, and possibly thestructure, of the YUNR motif in the loops of FinPmay play a smaller role than in other systems.37

There are two differences between the systemsanalysed for mutations in the YUNR motif so far.First, in hok-RNA, CopT, RNAI (ColE1), repZ-RNAand RNA-OUT the YUNR motif is located at pos-itions 1–4 of a 6 nt loop, whereas on the other

hand, it is located at positions 2–5 of a 9 nt loop inRNAII-L1 of pIP501 and in FinP (Loop I). Second, inhok/sok, CopT/CopA, repZ/IncI, and RNA-IN/OUT, the decisive interaction between sense andantisense RNA involves only one complementaryloop pair, whereas in RNAII/III of pIP501 and inFinP/traJ of F plasmid two complementary looppairs interact. Whereas in the first cases rather drasticeffects on inhibition in vitro and-CopA/CopT in vivo-were observed, Gubbins et al. and our groupdetected rather modest effects. One explanationcould be that, when two complementary loop pairscontribute to the interaction, only simultaneousmutations in both loop pairs would yield similareffects as those found for the one-loop-systemshok/sok or CopT/CopA. Whether or not thelocation of the U-turn motif within the loop is signifi-cant, can be only found after the analysis of moresystems and when in all cases the in vitro data aresupplemented with in vivo data.

Our former failure to observe inhibition orduplex formation with a truncated RNAIII speciescomprising only loop L3 (RNAIII47,

26) might, prob-ably, have been due to the requirement of both L3and L4 for efficient inhibition. In the case of sta-phylococcal plasmid pT181 where antisense RNAmediated transcriptional attenuation was discov-ered first38 our previous in vitro analysis demon-strated that both stem-loops of the 85 nt RNAI(which are identical with the two 50 stem-loops ofthe second antisense RNA, 144 nt RNAII) wererequired for pairing with the repC RNA.39 So, inboth well analysed cases of antisense RNAmediated transcriptional attenuation, two anti-sense RNA stem-loops seem to be required for effi-cient interaction with the target. Future work isdirected at the elucidation of the binding pathwayand the analysis of binding intermediates forpIP501 RNAII/RNAIII.

Materials and Methods

DNA preparation, manipulation and copy-number determination

Plasmid DNA was isolated from B. subtilis DB104.42

DNA manipulations like restriction enzyme cleavage andligation were carried out using the conditions specified bythe manufacturer or according to standard protocols.40 APCR kit from Roche was used for PCR amplifications.DNA sequencing was performed according to the dideoxychain termination method41 with a Sequenase kit fromAmersham Bioscience. Copy numbers of pIP501 deriva-tives in B. subtilis were determined as described23 exceptthat gel photographs were scanned and band intensitiesquantified using the PCBAS 2.0 program.

Construction of E. coli/B. subtilis shuttle vectorscontaining mutations in the sequence for the50-target RNA loop

Plasmid pPRC/1 (see Table 1 for all plasmids used inthis study) containing the wild-type control region, was


constructed in the following way: first, a Pst I site wascreated at position 58222 to facilitate the subsequent con-struction of mutations in the leader region: a PCRderived Bam HI/Pst I fragment spanning nt 160 till 582was inserted into the pUC19 Bam HI/Pst I vector result-ing in plasmid pUCU1. Furthermore, a PCR derivedPst I/Hind III fragment comprising nt 584 till 1623 wascloned into pUC19 Pst I/Hind III vector yielding plasmidpUCR1. The pUCR1 Pst I/Hind III fragment and thepPR1 Hind III/EcoR I fragment were cloned togetherinto the pUC Pst I/EcoR I vector resulting in pUCR3.The Pst I/Eco RI fragment of pUCR3 and the Bam HI/Pst I fragment of plasmid pUCU1 were jointly clonedinto the pPR4 Bam HI/Eco RI vector23 yielding plasmidpPRP. Subsequently, the copR gene was inserted as a549 bp Eco RI fragment derived from plasmidpCOP1B224 into the unique Eco RI site of plasmid pPRP,and the plasmid containing copR in the same directionas the repR gene was designated pPRC/1. All PCR gener-ated fragments were confirmed by sequencing.

All mutations in the leader region were obtained asfollows.

Mutated Bam HI/Pst I fragments were generated usingpPR1 as template and a two-step PCR with primer SB214(50 TAG AAG CTA CGA TCA AAG TTG AA) and muta-genic primer 1 on the one hand and primer 846-30 (50

GAA TTC GGA TCC AGA TCT AAC AGA ACC AGAACC AGA) and mutagenic primer 2 on the other handin the first step and 846-30 together with SB214 in thesecond step. After digestion with Bam HI and Pst I, theywere jointly cloned with the Pst I/EcoR I fragment ofplasmid pUCR3 into the pPR4 Bam HI/Eco RI vector.Afterwards, the copR gene was inserted as Eco RI frag-ment in the desired orientation as described above.

The following combinations of mutagenic primerswere used (primer 1 indicated above primer 2):

pPRC1/1 (U-A mutation)

SB193: 50 GAC CAGT TTA AAG CCA GAG AAA TTTTAA CTSB194: 50 AGT TAA AAT TTC TCT GGC TTT AAC TGGTC

pPRC3/1 (loop inversion, i.e. 3 0 YUNR)SB251: 50 GAG CCA CGA CCA GTT AAA GAA AGAGTC CTT TTA ACT GCG AGC CTT AASB252: 50 TTA AGG CTC GCA GTT AAA AGG ACT CTTTCT TTA ACT GGT CGT GGC TC

pPRC4/1 (silent mutation N-C)SB244: 50 GAC CAG TTA AAG CCT CAG AAA TTTTAA CTGSB245: 50 CAG TTA AAA TTT CTG AGG CTT TAA CTGGTC

pPRC5/1 (flip-flop-mutation)SB275: 50 GAG CCA CGA CCA GTT AAA GTT TCTCAG GCT TTA ACT GCG AGC CTT AASB276: 50 TTA AGG CTC GCA GTT AAA GCC TGAGAA ACT TTA ACT GGT CGT GGC TC

Construction of vectors expressing wild-type andmutant RNAIII species

PCR was performed on each of the plasmids pPRC1/1, pPRC2/1, pPRC4/1 and pPRC5/1 as templates witholigonucleotides SB354 (50 TCT GGG AAT CCA ATCAAC TAA GTTT TTC T) and SB355 (50 TCT AGA AAG

CTT AAC GAA CTG AAT AAA GAA TAC). The result-ing 260 bp fragment comprising rnaIII was cleaved withBamHI and HindIII and inserted into plasmid pGK14cleaved with the same pair of enzymes yielding plas-mids pGKUA, pGKLI, pGKNC and pGKFF, respectively.

In vitro transcription and secondarystructure analysis

RNAII and RNAIII were synthesized in vitro by run-off transcription with T7 RNA polymerase from PCR-generated DNA templates as described26. Labelled RNAspecies were purified from 6% or 8% sequencing gels.“Unlabelled” RNAIII or RNAII-species were synthesizedin the presence of 100 mM [3H]UTP to allow for accuratedetermination of the RNA concentration, and sub-sequently gel-purified. All radiochemicals were pur-chased from Hartmann-Analytic, Braunschweig.

Partial digestions of in vitro synthesized, 50-end-labelled RNAIII and RNAII species with ribonucleasesT1, T2 and V were carried out as described26 except thatafter five minutes digestion in a total volume of 5 ml,5 ml formamide loading dye were added, samples wereincubated for five minutes at 95 8C and aliquots (3–5 ml)were separated on an denaturing 8% polyacrylamidegel. Alkaline ladders were generated as described.26

Single-round transcription assays

Single-round transcription assays were performed asdescribed previously26 using PCR-generated 500 bpDNA-fragments of pPRC/1, pPRC1/1, pPRC3/1,pPRC4/1 or pPRC5/1 (comprising promoters pII andpIII, the attenuator and 100 bp downstream) as thetemplates. The transcription buffer contained 120 mMTris–HCl (pH 7.8), 25 mM MgCl2, and 2 mM DTT. In thepreincubation step, initiation complexes were allowedto form by the inclusion of 100 mM of the dinucleotideApA (Sigma), 20 mCi [a-32P]ATP (Hartmann-AnalyticBraunschweig), an NTP mix containing 1 mM ATP and20 mM CTP, template DNA (,1029 M) and B. subtilissA-RNA polymerase at <79 mg/ml (prepared by J. M.Sogo, Madrid). After five minutes at 30 8C, solution 2 wasadded to start elongation of the pre-formed, short RNAchains. Solution 2 contained, in addition to the transcrip-tion buffer, rifampicin at a final concentration of 10 mg/mland all four NTPs to yield final concentrations of 100 mM,to allow the elongation of single chains, and prevent newinitiations. Aliquots withdrawn at appropriate times werephenol-extracted, precipitated, dissolved in formamideloading dye, boiled and separated on 6% sequencing gels.Gels were dried, analysed in the PhosphorImager andquantitated as above. The protocol of the attenuationexperiments was as described.26

ENU (ethylnitrosourea) probing

ENU probing was done as follows: in vitro transcribedRNAII species were dephosphorylated and 50 labelledwith [g-32P]-ATP as described previously.26 The 50 end-labelled RNAs (20,000 cpm) were dissolved in a buffercontaining 150 mM sodium cacodylate, 5 mM MgCl2

and 5 mM KCl supplemented with 1 mg tRNA in a 15 mlreaction volume.

Subsequently, 5 ml of ethanol or saturated ENU sol-ution (in ethanol) were added and the 20 ml mix wasincubated at 37 8C for 30 minutes or at 90 8C for one min-ute, respectively. As a control, 5 ml of ethanol were added


instead of ENU, and the incubation was performed at37 8C for 30 minutes. Reactions were stopped by additionof ethanol and NH4-acetate, supplemented with 2 mgtRNA and subsequent centrifugation. Pellets werewashed twice with 80% (v/v) ethanol, resuspended in10 ml of 100 mM Tris–HCl (pH 9.0) and incubated forfive minutes at 50 8C for backbone scission. Backbonescission was stopped by addition of 10 ml of formamideloading dye followed by incubation for five minutes at95 8C and subsequent rapid cooling on ice. Five microli-tres were separated on a denaturing 8% polyacrylamidegel along with T1 and T2 digestions of the same labelledRNAs as markers. Band intensities were quantitatedusing the Fuji-PhosphorImager.

Acknowledgements

The authors thank Margarita Salas and J. Sogo,Madrid, for providing us with purified B. subtilisRNA polymerase. This work was supported bygrant Br1552/4-3 from the Deutsche Forschungsge-meinschaft (to S.B.).

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Edited by D. E. Draper

(Received 18 June 2003; received in revised form 10 September 2003; accepted 10 September 2003)


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Antisense-RNA Mediated Transcriptional Attenuation: Importance … · 2012-11-30 · Importance of a U-turn Loop Structure in the Target RNA of Plasmid pIP501 for Efﬁcient Inhibition