Proc. Natl. Acad. Sci. USAVol. 87, pp. 2675-2679, April 1990Biochemistry

Ribosomal protein L4 stimulates in vitro termination oftranscription at a NusA-dependent terminator in theS10 operon leader

(attenuation/autogenous control/Escherichia cola)

JANICE M. ZENGEL AND LASSE LINDAHLDepartment of Biology, University of Rochester, Rochester, NY 14627

Communicated by Charles Yanofsky, February 1, 1990

ABSTRACT The il-gene S10 ribosomal protein operon ofEscherchia coli is under the autogenous control of W, theproduct of the third gene of the operon. Ribosomal protein IAinhibits both transcription and translation of the operon. Ourin vivo studies indicated that IA regulates transcription bycausing premature termination within the untranslated S10operon leader. We have now used an in vitro transcriptionsystem to study the effect ofpurified IA on expression ofthe S10operon. We rind that the cell-free system reproduces the in vivoobservations. Namely, in the absence of IA, most of the RNApolymerases read through the termination site in the S10attenuator; the addition of IA results in increased terminationat this site. However, RNA polymerase does not terminate atthe S10 attenuator, with or without IA, unless an additionalfactor, protein NusA, is added to the transcription reaction.These results suggest that the attenuator in the S10 operon isa NusA-dependent terminator whose efficiency is regulated byribosomal protein IA.

The S10 operon of Escherichia coli contains the genes for 11ribosomal proteins (r-proteins). Expression of this operon isunder the autogenous control of r-protein L4, the product ofthe third gene (1-3). L4 regulates the S10 operon by inhibitingtranslation ofthe most proximal gene (3, 4) and by stimulatingpremature termination of transcription within the SlO leader(5). Genetic studies indicated that L4-mediated control oftranscription and of translation works by two independentmechanisms (4, 6).The site of L4-stimulated transcription termination is about

140 bases from the start oftranscription (5-7). This places thetermination site more than 30 bases upstream of the mostproximal structural gene, within a string of uridines on thedescending side of a stable hairpin structure (8). We haveproposed that this structure might function as a relativelyweak p-independent terminator that works more efficiently inthe presence of L4 (5, 6). To test this simple model and tobetter understand the molecular details of LA-mediated at-tenuation, we have studied the effect of purified r-protein L4on in vitro transcription of the S10 operon by using a simplecell-free transcription system. We find that L4 does indeedstimulate termination of transcription by RNA polymerase atthe same position in vitro as in vivo. However, attenuationrequires the addition of transcription factor NusA.

MATERIALS AND METHODSPlasmid Templates. Plasmids pLF1 (9) and pSma2 (7) are

shown in Fig. 1. Plasmid pLL226 (7), also shown in Fig. 1,contains the S10 promoter and leader followed by a partialtRNA gene (TDF1, see ref. 10) and a synthetic rrnC terminator

(11). Deletion derivatives of pLL226 were constructed bylinearizing the plasmid with Sma I, treating for various timeswith BAL-31 at 100C, digesting with Sty I, filling-in with theKlenow fragment ofDNA polymerase I, and religating (7).Chemicals and Enzymes. Uridine 5'-[a-[P5S]thio]tri-

phosphate (UTP[35S]) and [a-32PJUTP were from Amersham.Nonradioactive nucleoside triphosphates were from Pharma-cia or Sigma. RNA polymerase either was purified in ourlaboratory by standard procedures (12) or was a gift from T.Platt (University of Rochester) or E. Morgan (Roswell Park,Buffalo). p protein was from T. Platt, NusA was from T. Plattand from E. Morgan, factors NusB, NusG, and NusE werefrom J. Greenblatt (University of Toronto), and r-proteinswere from M. Nomura (University of California, Irvine) andfrom K. Nierhaus and P. Nowotny (Max Planck Institute,Berlin).In Vitro Transcription Reactions. The standard 20-Al reac-

tion mixture contained 40 mM Tris-HCl (pH 7.9), 10 mMMgCl2, 0.1 mM EDTA, 100-150 mM KCl, 0.2 mM dithio-threitol, 500 ,uM ATP, 500 ,tM CTP, 500 ,uM GTP, 100 ,uMUTP, 20-40 ,uCi of UTP[35S] (1 Ci = 37 GBq) or 12.5 puCi of[32P]UTP, and 1-2 pug of supercoiled plasmid DNA. RNApolymerase, r-protein L4 or S7, and other termination factorswere added at the indicated concentrations. All componentsexcept DNA were added on ice; the reaction was started byaddition of DNA. After 15 min at 370C, the reaction wasstopped by placing it on dry ice. Carrier RNA (10 ;kg of yeastRNA) was added, and total nucleic acids were extracted withphenol and then with chloroform/isoamyl alcohol [24:1 (vol/vol)], precipitated with ethanol, and resuspended in 5 ,ul of 10mM Tris-HCl, pH 7.9/0.1 mM EDTA. The RNA was mixedwith an equal volume of 95% (vol/vol) deionized formam-ide/10 mM EDTA/0.1% bromophenol blue/0.1% xylenecyanole and then fractionated on a standard DNA sequenc-ing gel. Where indicated, the RNA was first subjected to a T1nuclease mapping procedure (13) and then analyzed by gelelectrophoresis. In vivo-labeled RNA (2, 13) was analyzed byT1 nuclease mapping (7, 8, 13) in parallel with the in vitrotranscripts. RNA size markers were prepared as described(13).

RESULTSMapping of Attenuated and Read-Through Transcripts Syn-

thesized in a Purified Transcription System. We first analyzedthe effect of purified L4 on in vitro transcription of the S10operon leader by using supercoiled plasmid pLF1 DNA astemplate (Fig. 1) and a partially purified RNA polymerase.S10 leader RNA was then purified using a Ti nucleasemapping procedure (7, 8, 13) in which total transcribed RNAwas hybridized to a single-stranded DNA probe specific for

Abbreviations: r-protein, ribosomal protein; UTP[35S], uridine 5'-[a-[35S]thio]triphosphate.

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2676 Biochemistry: Zengel and Lindahl

AUT4 SD_

1 00 bases

S10 leader Sb' |acZ'

ATT* SD

S0 leader IlZAll* SD

:Sstl Smal Styl. 25B

* zzzzl 27B=oA///] 30B=,/i.ZZ 39B

toZZZZZZZ 26B,ZZZZZZZZZJ 7BEZZZZZZZZI 2Bvzizzzzzz] 56BLuz>,-/>> - 28BEZZZ{*/fZZZ3i lB

ih vivo in7 vitropLF1 pSma2 pLF1

~t 't st-J , X C/

+ + +

280 -*bases

150-*bases

- ATT'* ATT

FIG. 1. Maps ofS10 leader fusion plasmids. Plasmid pLF1 carriesthe beginning of the S10 operon, including the promoter (Ps10), a172-base S10 leader, and the proximal half of the structural gene forr-protein S10 fused in frame to lacZ'. Plasmid pSma2 contains thefirst 165 bases of the S10 leader upstream of an intact lacZ gene.Plasmid pLL226 also contains the first 165 bases of the S10 leader,placed upstream ofthe rrnC terminator (t). All three plasmids containan intact Shine-Dalgarno sequence (SD). The site of LA-stimulatedtermination in the leader (ATT) is indicated. The position of theleader hybridization probe used for T1 nuclease mapping is shownabove the pLF1 map. This probe (7) is an M13 derivative containing167 bases of S10 operon DNA from 2 bases upstream of thetranscription start site (8) to the Sst I site within the Shine-Dalgarnosequence ofthe S10 gene. Extents ofBAL-31 deletions in derivativesof pLL226 are shown by hatched bars below the pLL226 map.Relevant restriction enzyme sites are also indicated.

the S10 leader, nonhybridized RNA was digested with nu-clease T1, and the resulting protected RNAs were analyzedby gel electrophoresis. For comparison, we also analyzedRNA synthesized in vivo in the absence or presence ofexcessLA. The leader probe protected three major bands with the invitro RNA, two ofwhich migrated to the same position on thegel as the bands observed from in vivo-labeled RNAs (Fig. 2).The upper band corresponds to read-through transcripts-that is, RNA transcribed through the S10 attenuator regioninto the structural gene and trimmed back to the size of theleader probe by the nuclease. The lower band (actually adoublet band) corresponds to attenuated transcripts-that is,RNA terminated about 140 bases into the S10 leader, here-after referred to as ATT transcripts. Although not shown, themobility of this doublet was not altered when the transcriptswere purified in the absence ofT1 nuclease, as expected iftheRNA were contained entirely within the sequence carried onthe probe. From these results we conclude that the S10attenuator functions in vitro. Moreover, the attenuator re-sponds as expected to L: the addition of this protein resultedin an increase in the level of attenuated transcripts and adecrease (visible in shorter exposures than the autoradiogramshown in Fig. 2) in the level of read-through transcripts.Other r-proteins, including S7 (Fig. 2), L11, and S4 (data notshown) had no effect on attenuation.The only discrepancy between the in vitro and in vivo

results was that in the cell-free system there was a significantamount of transcription termination just beyond the attenu-ator, generating transcripts (actually a series of three bands)that were not observed in the in vivo samples and thathereafter will be referred to as ATT' transcripts (see Fig. 2).The sizes of these ATT' RNAs correspond to terminationwithin a series of three uridine residues just downstream ofthe four uridines at which the LA-stimulated attenuation

FIG. 2. Mapping of in vivo and in vitro transcripts from the S10operon. In vivo transcripts were from cells carrying plasmid pLF1 orpSma2 pulse-labeled for 3 min with [32P]phosphate before (lanes -)or 10 min after (lanes +L4) induction of LA oversynthesis from asecond plasmid, pLF17 (9). In vitro transcripts were synthesized ina 20-Al reaction mixture containing 2 Etg of RNA polymerase(partially purified; see text), 2 ,ug of supercoiled plasmid DNA, 12.5ILCi of [32P]UTP, and 0.9 i&g of LA, 0.6 ,.g of S7, or the equivalentvolume of protein buffer. Transcripts were hybridized to a leaderprobe (Fig. 1) and digested with T1 nuclease. Protected RNAmolecules were then fractionated on a denaturing urea/polyacryl-amide gel (13). Positions of size markers are indicated on the left.Bands corresponding to read-through (RT) and attenuated (AUT and,for in vitro samples, AUT') transcripts are indicated on the right.

occurs (Fig. 3). The relevance ofthese RNAs is not clear, but,as shown below, termination resulting in ATT' transcripts isaffected by the source ofRNA polymerase and, under certainconditions, is also stimulated by protein U.

Construction of Plasmid pLL226. To analyze L4-stimulatedtermination more quantitatively, we constructed plasmidpLL226, containing the first 165 bases of the S10 leader fusedto a strong terminator from the rrnC rRNA transcription unit(Figs. 1 and 3). This template allowed us to detect read-throughtranscripts without the T1 nuclease mapping procedure, sincemost of the transcripts extended beyond the attenuator ter-minated at the rrnC terminator, generating read-through mol-ecules about 285 bases long. Plasmid pLL226 is under normalin vivo transcription regulation by LA (7).The results of a typical transcription reaction with pLL226

are shown in Fig. 4, lanes pLL226. Total RNA synthesizedin the cell-free system was fractionated on the gel. The majorspecies ofRNA correspond to transcripts initiated at the S10promoter and terminating either at the rrnC terminator (read-through RNA) or at the attenuator (AUT and AUT' RNAs).Again, the addition of purified L4 stimulated termination atthe S10 attenuator, resulting in a decreased level of read-through RNAs and an increased level of attenuated tran-scripts.

Determination of the 3' End Requirements for Attenuationin the S10 Leader. r-Protein L4 regulates not only transcrip-tion but also translation of the S10 operon. Our in vitrotranscription experiments show that ribosomes are not re-quired for L4 regulation of the S10 attenuator. Nevertheless,we wanted to eliminate from plasmid pLL226 the distalregion of the leader to confirm that sequences involved ininitiation oftranslation of the proximal gene ofthe operon arenot required for transcription control. We were also inter-ested in determining more precisely the 3' end requirementsfor the S10 attenuator. For these reasons, we isolated deriv-

Probe:

pLF1 Ado

pSma2 PSIO

pLL226 Ps10-U-f

0

0

0in

co-J

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Attenuator hairpin

A 1009CG

Ac AU AUGUA *-1B UAAU. UACG GUC G 28B GCCG

C

UG AU

UA

Cu c]56BG4U'] - ATTCG

A

UA A GCG UG GA A C U

100 SCG|4. UA CAU U UAAUACC

UACG7B+UA

150~GU

AU GC 26BA U C A

AG G U A 4-26BaU U CG AUG U CG GUCG GC .AuJ 4- ATUA AU AU4B

UUAUA

GC 39B 30B1 27B 25BGC CGU'A

GC* C AC U U UAUAAAAUAATTGGAGCUCgguacccggggaucc...PPP G CAU GC 0 I

GC CG 150

GC UA Sstuscte Sma IsiteUA §CGUUA C

CGCG ATT'

A Cs A CU U

FIG. 3. Secondary structure of the 510 leader. Determination of the secondary structure has been described (8). Bases from the S10 leaderare in uppercase letters; bases from the plasmid carrying the rrnC terminator are in lowercase letters. The hairpin structure involved in LAregulation of transcription is indicated. We have not directly determined the secondary structure of the pLL226 transcript, but we assume theleader structure through the first 150 bases is unchanged. The rest of the sequence is shown as unstructured RNA. Termination sites within theS10 leader (ATT and ATT') are indicated; major sites follow the boldfaced Us. Endpoints of BAL-31 deletions are also indicated. TheShine-Dalgarno sequence for the S1O gene (GGAG) and pertinent restriction sites are indicated. Sequences that are also found in 23S rRNAat the proposed L4 binding site include bases 117-125, 146-150, 152-155, and 159-163. (Inset) Lower half of the attenuator hairpin of deletionmutant 26B. The sequence downstream of the deletion is indicated by boldfaced letters.

atives of pLL226 deleted for various extents at the 3' end ofthe leader (Figs. 1 and 3). The results of transcriptionreactions with these mutant templates are shown in Fig. 4.Deletion mutants 25B, 27B, 44B, 30B, and 39B, all containingthe intact attenuator hairpin (Fig. 3), gave results indistin-guishable from the results with the wild-type parent plasmidpLL226. That is, addition of protein L4 resulted in anincreased level of attenuated transcript and a decreased levelof read-through transcript. Deletion mutants 2B, 7B, 56B,1B, and 28B, which lack portions of the attenuator hairpin,showed no prematurely terminated transcripts, indicatingthat these mutants lack sequences necessary for attenuation.Mutant 26B gave intermediate results (see below).

Quantitation of L4-Stimulated Termination of Transcrip-tion. To quantitate the effect ofL4 on the various derivativesof pLL226, the amount of radioactivity in the read-throughand attenuated RNAs was measured. The results are sum-marized in Table 1. All templates containing an intact atten-uator hairpin (pLL226 through 39B) showed a basal level oftermination that generated ATT RNA at 10-15% of totalRNA. Addition of L4 stimulated attenuation 2- to 3-fold,concurrent with a decrease in the amount of read-throughtranscripts. L4 had no measurable effect on termination thatgenerated ATT' RNA. These in vitro results suggest that theproximal 149 bases ofthe S10 leader contained in mutant 39Bare sufficient for L4-stimulated termination of transcription,consistent with our in vivo analysis of this mutant (7).Mutant 26B showed a very low basal level of termination

that generated ATT RNA (Table 1) as well as an altered band

pattern in that region of the gel (Fig. 4). Nevertheless,addition of L4 led to increased transcription termination inthe region of the attenuator. These results suggest that theterminator is weakened by a deletion extending up to base 139of the leader but has residual function and can still respondto L4. These results may be explained by the fact that thedeletion in mutant 26B brings in a downstream DNA se-quence that restores the two internal uridines in the 4-baseuridine string at the attenuation point (see Fig. 3 Inset).

Deletions extending further upstream of the L4-stimulatedtermination site, which partially or completely destroy theattenuator hairpin, abolished the attenuator function. Theamount of radioactivity in read-through RNAs was not sig-nificantly decreased after addition of L4 to reactions con-taining mutants 2B, 7B, 56B, 1B, or 28B (data not shown).However, we were surprised that IA still had a subtle butreproducible effect on these deletion plasmids. Namely, inthe presence of L4 there was a small change in the pattern ofbands corresponding to the read-through transcripts termi-nating at the rrnC terminator, such that there was an increasein the intensity of the lower band (Fig. 4). This effect was notobserved with deletions leaving a functional attenuator.NusA Protein Is Necessary for in Vitro Attenuation. Since

control of transcription termination often involves auxiliaryproteins, we wanted to determine if any known transcriptionfactors could influence the efficiency (and possibly the pre-cise site) oftermination in vitro. Our initial experiments wereperformed with a partially purified RNA polymerase. Wefound that with a more highly purified RNA polymerase

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2678 Biochemistry: Zengel and Lindahl

DNA:

L4:

RT-

cD BAL-31 deletion plasmids

n cmcom m m m m ml-j 10 ,-. '4 0) 0) wcDf En mCO c0. C) N . c C C C'.J cN 10O- Co

-±+-+ -±-++ -+-+-+-+-++ -+

4-471

-377

-284

-210

4-150ATTLATT--

FIG. 4. Effect of LA on in vitro transcription from plasmidpLL226 and its BAL-31 deletion derivatives. The transcriptionreaction mixtures were as described in Fig. 2, except each mixturecontained 4 A&g of partially purified RNA polymerase, 30 ,uCi ofUTP[35S], and 0.25 ,ug of L4 or the equivalent volume of proteinbuffer. Positions of read-through (RT) and attenuated (ATT andATT') transcripts are shown on the left. Positions of size markers areindicated on the right. Read-through RNA from mutant 7B migratesfaster than RNAs from mutants with similarly sized leaders becausethe deletion in this mutant extends about 20 bases downstream of theSty I site that marks the 3' boundary ofdeletions in the other mutants(Fig. 1).

preparation, there was no termination at the S10 attenuator,even in the presence of L4, unless NusA protein (14) wasadded (Fig. 5). Under the same reaction conditions, otherNus factors, including NusB (15, 16), NusG (17), and NuOE(18, 19), had no significant effect on transcription of the S10leader (Fig. 5). Termination factor p (20) also had no effect(data not shown). These results suggest that the attenuator inthe S10 operon is a NusA-dependent terminator whoseefficiency is stimulated by r-protein L4.

Since in our initial transcription experiments (Figs. 2 and4) we observed L4-mediated attenuation without addition ofNusA, we surmised that our original RNA polymerase prep-aration might contain NusA. Gel electrophoretic analysisconfirmed that this RNA polymerase is contaminated with asmall amount of other proteins, including NusA (data notshown). We conclude that there is a sufficient amount ofNusA to mask the requirement for this factor. A stableassociation between RNA polymerase and NusA during thepurification of the enzyme has been reported (21).

In the transcription reactions with the more highly purifiedRNA polymerase, we observed less termination that gener-ated ATT' transcript than we had observed with the originalRNA polymerase (compare Fig. 4 with Fig. 5). Moreover, theAlT' transcripts exhibited the same pattern as the AlT

Table 1. Quantitation of the effect of r-protein L4 onin vitro attenuation

% total RNAs

DNA L4 AlT ATT' RTpLL226 - 10 32 59

+ 33 33 34+/- 3.3 1.0 0.58

25B - 11 29 60+ 32 29 39

+/- 2.9 1.0 0.6527B - 14 34 52

+ 30 32 38+/- 2.1 0.94 0.73

44B - 10 36 54+ 34 34 32

+/- 3.4 0.94 0.5930B - 9 31 60

+ 28 31 41+/- 3.1 1.0 0.68

39B - 13 26 61+ 29 27 45

+/- 2.2 1.0 0.7426B - 4 8 88

+ 15 6 79+/- 3.8 0.75 0.90

Radioactivity in the bands corresponding to attenuated (AUT andATT') RNAs and read-through (RT) RNAs terminating at the rrnCterminator was determined (1) from the gel shown in Fig. 4. Valuesfor ATT and AlT' RNAs were corrected for background by sub-tracting the cpm in the corresponding areas of the gel from samplesnot terminating at the attenuator. These corrected values and theuncorrected values for read-through RNAs were then normalized tothe number ofuridines in the transcript (38 for AlT, 41 for AlT', and49-76 for read-through transcripts). Each nofinalized value was thendivided by the total normalized cpm (ATT plus ATT' plus RT) togenerate the percent ofRNA polymerases terminating at a given siteto yield the indicated RNAs. The effect of L4 was calculated bydividing the value in the + L4 reaction by the value in the - L4reaction (+/-). Addition of L4 had no significant effect on the totalnumber of transcripts from a given template (data not shown).

transcripts: stimulation by L4 but only in the presence ofNusA. We cannot yet account for these polymerase-dependent variations in termination that generates ATT'transcripts, nor can we explain why these transcripts are not

NusE(S10) + - + - + I- 1-NusB& NusG + - + -

NusA +L4 + + + + + + +I+

RT-4

ATT'_-so wATT_*

FIG. 5. Effects ofNus factors on attenuation. Each 20-/.l reactionmixture contains 0.18 lug (20 nM) of purified RNA polymerase, 2 ,ug(50 nM) of 39B DNA, 20 pCi of UTP[35S], and, where indicated, 80nM NusB, 80 nM NusG, 80 nM NusE, and 160 nM L4 (+) or S7 (-).The positions of read-through (RT) and attenuated (ATT and ATT')transcripts are indicated.

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detected with in vivo-synthesized RNA. Perhaps ATT' tran-scripts are also formed in vivo but are rapidly shortened bynucleases to the same size as ATT molecules.

DISCUSSIONThe S10 operon attenuator functions as a terminator in asimple cell-free transcription system containing RNA poly-merase and transcription factor NusA. Furthermore, thelevel of termination is increased by the addition of r-proteinL4. The site of termination and the stimulatory effect of L4are consistent with our in vivo data showing that oversyn-thesis of r-protein L4 leads to an increased level ofprematuretermination at the S10 attenuator (5, 7). The relatively simplein vitro requirements for attenuator function together with theresults using the BAL-31 leader-deletion mutants confirm ourconclusion from in vivo genetic and physiological studies thattranscription control by L4 is achieved by a mechanism thatis independent of the inhibition of translation by L4. Eventhough there is excellent agreement between the in vitro andin vivo experiments, it should be pointed out that the in vitroexperiments cannot distinguish between very strong pausingat the attenuator and actual release of the transcript.The nusA gene product was originally identified as a factor

required for N-dependent antitermination in bacteriophage A(14). It has since been shown in a variety of transcriptionsystems that NusA enhances RNA polymerase pausing and,depending on the transcription unit, causes an increase or adecrease in termination (for review, see ref. 22). Thesecomplex effects of NusA suggest it may function as atranscription "fidelity" factor, interacting with RNA poly-merase and other termination factors to modulate the enzymeresponse to pause and termination sites (23). Although NusAhas not been shown to be absolutely essential for in vivoregulation of any bacterial gene, our in vitro experimentssuggest that NusA is required for termination (or a very stablepause in elongation) at the S10 attenuator and, therefore, isa necessary cofactor in L4-mediated regulation of transcrip-tion of the S10 operon. Several lines of evidence suggest thatthe boxA sequence, CGCTCTTA, is involved in NusA reg-ulation of A gene expression (24, 25). Even though similarsequences have been found in other operons whose tran-scription is affected by NusA, including rRNA transcriptionunits (26, 27), the precise role of boxA-like sequences inNusA function is still not clear. In fact, in some cases boxAseems to be dispensable for NusA activity (see, e.g., refs.28-31). The S10 leader has no boxA sequence, although itdoes have a sequence, AACAAT (bases 61-66; Fig. 3), whichis homologous to the recently extended portion ofboxA (32).Whether that sequence plays any role in NusA regulation ofthe S10 operon needs to be determined.The BAL-31 deletion analysis suggested that, in the ab-

sence of a functional attenuator, L4 still affected terminationof transcription, but at a terminator further downstream. Thestimulation of attenuation and the effect at the rrnC termi-nator seem to be mutually exclusive, since we observed thedownstream effect only when the deletion abolished attenu-ation. One model to explain these results is that r-protein L4programs a fraction of the RNA polymerase molecules toterminate at the S10 attenuator. If the attenuator is defectiveor deleted, the same modified RNA polymerases respond ina different way to the rrnC terminator. This model needs tobe tested experimentally, but it does raise two interestingpossibilities. (i) The target for L4 may lie upstream of thedeletion endpoint in mutant 1B, that is, upstream of base 119.This implies that L4 recognition does not require the hairpinstructure involved in attenuation or the leader sequenceshomologous to sequences in the L4 binding domain of 23SrRNA (see Fig. 3), which were originally thought to benecessary for L4 control (6, 33). (ii) r-Protein L4 can mod-

ulate termination at not only the S10 attenuator but also therrnC terminator. Whether this effect is simply an artifactresulting from the fusion of the S10 leader to a foreignterminator or an indication that L4 plays a more general rolein regulating transcription of other operons in E. coli remainsto be seen. In any event, it is interesting that a singler-protein, L4, plays three important roles in the cell: as acomponent ofthe 50S subunit ofthe ribosome, as aregulatoryprotein causing decreased translation of its own transcript,and as a termination factor regulating transcription of its ownoperon.

We thank T. Platt, M. Nomura, J. Greenblatt, K. Nierhaus, P.Nowotny, and E. Morgan for generous gifts of proteins, and W.McClain for the TDF1 plasmid. We also thank E. Grayhack forcritical reading of this manuscript. This research was supported bya grant to L.L. from the National Institute of Allergy and InfectiousDiseases.

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