Proc. Nadl. Acad. Sci. USAVol. 91, pp. 7543-7547, August 1994Biochemistry

Reduced Rho-dependent transcription termination permitsNusA-independent growth of Escherichia coli

(PatY/tansrption-trauon coupling)

CHUANHAI ZHENG AND DAVID I. FRIEDMAN*Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109-0620

Communicated by Michael J. Chamberlin, April 8, 1994

ABSTRACT NusA and Rho are essential Escherichia coilproteins that influence tanscription elongation and termina-tion. We show that an E. coli derivative unable to expressNusA, because its sole nusA gene contains a lrge dele-tion/substitution, is viable providing that the bacterium alsocarries a rho mutation that reduces tra iption termination.This Rho-miated suppression is not allee specific, sinceeither a mutation changing amino acid 134 [rho(E134D)] or amutation canging amino add 352 (rho)) allows growth of anusA-deleted E. coil. However, both rho mutations similarlydecrease transcription termination 8- to 9-fold. We proposethat the essential role of NusA is to enhance pausing of RNApolymerase at certain sites, permitting tight coupling of tran-scription and translation. This coupling interferes with Rhoaccess to and/or movement on the nascent RNA and blockspremature termination of transcription. Thus, NusA-dependent coupling should be less important in a mutant withlow Rho activity. The fact that E. coi grows without NusAargues that NusA should be considered an accessory factorrather than a subunit of RNA polymerase.

Transcription elongation can be considered to include allevents in transcription that follow initiation and precedetermination. While the factors regulating initiation have beenrelatively well characterized, those controlling elongationand termination are not as well understood. In fact, thecharacteristics of the elongation complex are currently mat-ters of some contention (1-4). For example, there is dis-agreement as to the size of the DNA-RNA hybrid within thetranscription bubble as well as the physical state of thepolymerase itself (4, 5). In addition to the components ofcoreRNA polymerase, five proteins, the products of the nusA,rho, greA, greB, and nusG genes (6-8), have been found toinfluence transcription elongation and termination. A sixthfactor, Tau protein, has not been genetically characterized,but it has been implicated in termination (9).Roberts (10) identified Rho based on its ability to terminate

transcription initiating from the early phage promoters PL andPR. Under certain conditions, Rho also terminates transcrip-tion within genes (11). Transcriptional polarity, observed inprokaryotes where transcription and translation are coupled,appears to follow from cessation of translation that uncoversRho-dependent transcription termination signals in the na-scent RNA, leading to the premature termination of tran-scription (12). NusA was initially identified because of its rolein the read-through of terminators mediated by the N proteinof phage A (13). NusA, also called L factor, was shown tostimulate 3-galactosidase synthesis in an in vitro coupledtranscription-translation system (14). Subsequently, in vitrotranscription studies showed that NusA enhances pausing ofRNA polymerase and influences transcription termination

(11, 15-19). Ward and Gottesman (20), by showing that thenusAl mutation reduces the effect ofsome polar mutations inbacterial genes, implicated NusA in the process of polarity.The Escherichia coli nus genes, identified by the isolation

of mutations that cause a failure in A N-mediated transcrip-tion antitermination (6, 17, 21, 22), encode products thatserve important functions for the E. coli host. Three muta-tions influencing the action of nus gene products are impor-tant to this study: nusAl identified NusA as a transcriptionfactor (13); nusE71 is an allele of rpsJ, encoding ribosomalprotein S10 (23); and rpoA(D305E) is a mutation in the rpoAgene that suppresses the effect of either the nusAl or thenusE71 mutation (A. T. Schauer, S.-w. C. Cheng, C.Z., L.St. Pierre, and D.I.F., unpublished work).The isolation of a number of conditionally defective nusA

mutations that cause loss of bacterial viability indicates thatNusA is an essential function (24-27). In vitro studies showedthat NusA binds to core RNA polymerase but not to holo-enzyme. Thus, NusA is thought to access RNA polymeraseafter sigma is released and stay associated with the polymer-ase throughout the elongation process (28, 29). According tothis scenario, it might be concluded that NusA is an essentialcomponent of RNA polymerase. The numerous studiesshowing functional interactions between Rho and NusAsuggest that some form ofinteraction between NusA and Rhomight explain the lethal effects ofnusA mutations. Consistentwith such an argument, we report the isolation of therho(E134D) mutation that allows E. coli to grow in theabsence of NusA.

MATERIALS AND METHODSMedia, Bacteria, Phage, and Genetic Techniques. The me-

dia used in these studies were prepared as described (30). Thebacterial strains used for this study as well as their relevantgenotypes are as follows: AB1133 (13); K37, galK> (30);K4012 (derived from K37), nusAll (24); YN3206,nusAamll3 supF6(Ts) (26); 333, trpE9851 rho) (suAl) (31);K4195 (derived from AB1133), nusAl rpsJ-nusE71rpoA(D305E); K4198 (derived from K4195), nusAl rpsJ-nusE71 rpoA(D305E) rho(E134D); K7314 (derived fromK37), rho(E134D); K7131 (derived from YN3206),nusAamll3 supF6(Ts) rho(E134D); K7619 (derived fromK37), AnusA533::cam rho(E134D); K7487 (derived fromK37), rho). The specialized transducing phage, AcI857 nusA(Aatt), was obtained from S. Adhya (National Institutes ofHealth).

Standard procedures were followed for both transforma-tion of DNA (32) and P1 transduction (33). Plasmids weredescribed in the indicated references or constructed bystandard cloning techniques (32): pMAK108 (34), pKL600(35), pKLtR3 (36), pNAG15 (30), pNAG3 (24), pNAG3-AnusA533::cam (this work). Enzymes and buffers were used

*To whom reprint requests should be addressed.

7543

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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according to manufacturers' instructions and were purchasedfrom New England Biolabs, Boehringer Mannheim, Be-thesda Research Laboratories, and Promega.

Cloning ofthe rho(E134D) and rhol Alleles. The rho(E134D)allele was cloned on a 6.7-kb HindIII fragment from E. colistrain K4198 in pBR325. The appropriate clone was identifiedby colony hybridization (32) using the 3-kb Pvu II/HindIIIagment from pMAK108 (34) as a probe. The rho) allele wascloned by the PCR. The reaction mixture consisted of 0.1 pgof chromosomal DNA from strain 333 (rho)) and 100 pmol ofthe primers 5'-GTTTTCCCGGTACCGGTTTG-3' (sensestrand 5' to rho) and 5'-CCCAAGCTTCCTAACATGCCC-AGCGCG-3' (antisense strand 3' to rho). Taq DNA poly-merase (2.5 units) (Boehringer Mannheim) was added to thePCR buffer (50 mM KCI/10 mM Tris HCl/1.5 mM MgCl2/0.01% gelatin) and reactions were carried out in a thermo-cycler for 20 cycles.Marker Rescue, Galactokinase Assay, DNA Sequencing, and

Western Blot Analysis. Marker rescue and galactokinaseassay were performed as described (24, 37). The dideoxynu-cleotide sequencing system Sequenase version 2.0 (UnitedStates Biochemical) was employed using dATP[a-35S] (Am-ersham). Both strands of the DNA were sequenced. Westernblot analyses with polyclonal rabbit antisera were performedessentially as described (30).

Construction ofE. coli with AnusA533::cwn. (i) Constructionof plasmid pNAG3-AnusA533::cam. A 0.5-kb Bcl I/Nco Ifiagment was removed from the nusA coding region ofplasmidpNAG3 (24) and replaced with the 1.3-kb BstUI fiagment frompBR325 containing the cam' gene. A construct, pNAG3-AnusA533::cam, with the camr gene positioned so that it istranscribed in the same direction as the nusA gene, wasidentified. (ii) Construction of an E. coli strain with theAnusA53::cam allele. Phage AcI857AnusA533::cam was con-structed by replacing the nusA allele of AcI857nusA(Aatt) withthe AnusA533::cam cassette through homologous recombina-tion with plasmid pNAG3-AnusA533::cam. E. coli derivativescontaining AnusA533::cam as the only nusA allele were con-structed as described by Irani et al. (38) (Fig. 1).PCR Analysis of Chromosomal nusA Alleles. Two primers

matching opposite strands of the DNA sequences locatedwithin the nusA coding region and 939 nucleotides apart weresynthesized. The sequence ofthe 5' primer, at nucleotide 301of the nusA gene, is 5'-ACCTTTGACCGTATCAC-3' (an-tisense strand) and that ofthe 3' primer, at nucleotide 1239 ofthe nusA gene, is 5'-TCGCGCAGTGCTTCAACGGTC-3'(sense strand). PCRs were carried out as described above.

Ecoli

RESULTSIdentfication of the rho(E134D) Mutation. We used a ge-

netic approach to identify possible functional interactions ofNus proteins with other E. coli encoded proteins. Towardthis end, K4195, a strain carrying three mutations known toinfluence A N-mediated antitermination [nusAl, rpsJ-nusE71, and rpoA(D30SE)], was constructed. Physiologicalstudies revealed that this triple mutant is thermosensitive.To identify the physiological defect resulting from the

combination of the three mutations, K4198, a mutant ofK4195 that grows at 420C, was characterized. On the basis ofresults of Hfr crosses and P1 transductions, the additionalsuppressor mutation in K4198, conferring the ability to growat high temperature, was located at minute 84.8 on the E. coligenetic map, closely linked to rhL8 (39). Complementationand marker rescue experiments located the suppressor mu-tation in a 0.9-kb BamHI/Hpa I fiagment from K4198 withinthe open reading frame of the rho gene.TheDNA sequence ofthis 0.9-kb fragment was determined

and compared to the corresponding wild-type sequence. Asingle base pair change was identified, which results insubstitution of an aspartic acid codon (GAT) for a glutamicacid codon (GAG) at codon position 134 in the rho gene.Based on this sequence change, we have named the sup-pressing mutation rho(E134D). The rho(E134D) allele byitself has no apparent effect on cell growth (data not shown).

Termination Activity of rho(E134D). To determine whetherthe rho(E)34D) mutation influences transcription termina-tion, we used plasmid pKLtR3, which has the tR3 Rho-dependent terminator from phage A placed between the lacpromoter and a gaiK reporter gene (36). As shown in Fig. 2,if the bacterium has the rho+ allele, as in the case of K37,there is only a 3-4% read-through of the tR3 terminator.However, ifthe bacterium has the rho(E134D) allele, as in thecase of K7314, read-through is 25-35%. Thus, therho(E134D) mutation reduces Rho-dependent termination 8-to 9-fold.

Effect of rho(E134D) on Conditional nusA Mutants. Therho(E134D) mutation also suppresses the partial thermosen-sitivity imposed by the combination of the nusAl andrpoA(D30SE) mutations (data not shown). The observationthat the combination of the nusAl and rpoA(D305E) muta-tions caused thermosensitivity while other pairwise combi-nations of the three mutations in K4195 failed to do so (datanot shown) suggested that the nusAl mutation might be thesignificant contributor to the thermosensitivity of the K4195triple mutant. Therefore, we tested whether rho(E134D)

nusA

cI57AnusA533::cam

Integration throughhomologous recombination

a fblysogen

Excision throughhomologous recombination a/ of b

n sA

nusAins53 Hi

Ainu~sA 533. :canm

FIG. 1. Strategy for crossing AnusA533::cam from phage AcI857AnusAS33::cam to the E. coli chromosome. Stippled box, nusA gene; solidbox, cam' gene from pBR325; thick line, phage DNA; double thin lines, E. coli DNA; crossed lines, possible cross-over points for homologousrecombination.

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m 100-0

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.2 60-0.

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FIG. 2. Comparison of transcription termination by different rhoalleles. The isogenic E. coli strains used in this study, with their rhoalleles in brackets, are K37 [rho+ (solid bars)], K7314 [rho(E134D)(stippled bars)], and K7487 [rho) (hatched bars)]. Termination wasmeasured in vivo using plasmid pKLtR3. When the plasmid is in agaIK- bacterium, the amount of galactokinase expressed will varydepending on the level of read-through of the tR3 terminator. Thelevels of galactokinase present in extracts from the various E. coliderivatives containing the indicated plasmids (listed on the x axis) arerecorded as percentage of wild-type expression (listed on the y axis).In a representative experiment, pKL600, the parental plasmid ofpKLtR3, expresses 470 units of galactokinase activity in a rho+background. This figure represents results of three independentexperiments and error bars indicate deviation from the mean per-centage.

suppresses the growth defects of thermosensitive nusA mu-tations.K4012 fails to grow at temperatures above 410(, because

it has the missense nusAll(Ts) mutation (25). A derivative ofK4012 containing, in addition to nusAll(Ts), the rho(E134D)mutation is not thermosensitive. Although this result showsthat rho(E134D) suppresses the thermosensitivity caused bya mutation in nusA, the mechanism of this suppression is notobvious. Two mechanisms seem plausible: (i) rho(E134D)might foster NusA-independent growth or (ii) an allele-specific interaction might restore a protein-protein interac-tion between mutant NusA and mutant Rho at high temper-ature. Since the nusAl and nusAl) mutations change nearbyamino acids (25), the data presented so far do not allow us todistinguish between these two possibilities.We therefore tested whether the rho(E134D) mutation

suppresses thermosensitivity resulting from a qualitativelydifferent nusA defect, a nusA amber mutation. A bacteriumwith the nusAamll3 mutation must also have an ambersuppressor to be viable (26). E. coil strain YN3206 carries thenusAamll3 mutation with the supF6(Ts) allele. The amber-suppressing supF6(Ts) is not functional at higher tempera-tures (420() and therefore, because sufficient NusA is notexpressed at high temperature, growth of YN3206 is ther-mosensitive (26).The rho(E134D) mutation was moved into YN3206 by P1

transduction, creating strain K7131, which grows at 420(.This indicates that rho(E134D) suppresses nusAamll3, andtherefore rho(E134D) is not an allele-specific suppressor ofnusA mutations.

Levels of NusA Protein in the nusAamll3 supF6(Ts)rho(E134D) Strain. Western blots were used to determine thenature and quantity of NusA protein expressed in thenusAamll3 supF6(Ts) rho(E134D) strain K7131. The resultsare shown in Fig. 3A. The levels ofRho protein are similar inall lanes. Equivalent amounts ofNusA protein are expressed

AL B

-w .N IiS.-\usA

-t,1_ _ _ _

FIG. 3. Immunoblot analysis of NusA expression. Approxi-mately equivalent amounts of protein extracts isolated from expo-nentially growing cells were separated on a SDS/WM% polyacryl-amide gel and transferred to nitrocellulose. The nitrocellulose washybridized with anti-NusA and anti-Rho polyclonal antisera. Posi-tions ofNusA and Rho are indicated. (A) Lanes 1 and 3, extracts fromstrain K7314 [nusA+ rho(E134D)]; lanes 2 and 4, extracts from strainK7131 [nusAamll3 supF6(Ts) rho(E134D)]. Bacteria were grown at320C (lanes 1 and 2) or at 420C (lanes 3 and 4). (B) Lane 1, extractfromstrain K7314 [nusA+ rho(E134D)]; lane 2, extract from strain K7623(nusA+ rho+ and a camr marker at an unknown position); lanes 3 and4, extracts from strains K7619 and K7620, respectively [both areAnusA533::cam rho(E134D)]. Bacteria were grown at 37C.

by the nusA+rho(E134D) strain K7314 at 320C (lane 1) or at420C (lane 3). The nusAamll3 supF6(Ts) rho(E134D) strainK7131 expresses very low levels of NusA protein at 420C(lane 4) and almost normal levels at 320C (lane 2).The results with the nusAamll3 mutation are consistent

with the argument that suppression by the rho(E134D) mu-tation of nusA mutations that exhibit a thermosensitivephenotype is not allele specific. However, these experimentsdo not rule out the possibility that the small amount ofresidual NusA observed in the Western blot, perhaps result-ing from increased "leakiness" caused by the rho(E134D)mutation, is sufficient to support bacterial growth.

Construction of an E. coli Defective for NusA. To determinewhether E. coil can survive in the total absence of NusA, aderivative of a AnusA specialized transducing phage,AcI857AnusA533::cam, that has a defective nusA allele (theAnusA533::cam allele) was used to construct an E. coil strainlacking any NusA activity. E. coli lysogen withAcI857AnusA533::cam integrated at the nusA locus is thusdiploid for nusA+ and AnusA533::cam (see Materials andMethods and Fig. 1 for details). E. coli containing only oneof the nusA alleles were selected by plating lysogens on LBplates at 420( (38). Analysis ofcolonies cured ofthe prophagerevealed that 1 of every 5 bacteria was chloramphenicolresistant when the starting bacterium carried the rho(E134D)mutation, and only 1 of =50O bacteria analyzed was chlor-amphenicol resistant when the starting bacterium was rho+.The PCR was used to confirm that the putative NusA-

deficient strain indeed has the AnusA53::cam and no othernusA allele. Three of the 50 chloramphenicol-resistant colo-nies isolated in the selection starting with a bacterium car-rying the rho(E134D) mutation (K7619, K7621, and K7622)were randomly chosen for examination. All three show thePCR profile expected for the AnusA533::cam allele (Fig. 4,lanes 4-6). On the other hand, the only chloramphenicol-resistant colony isolated in the selection starting with abacterium carrying the rho+ allele K7623 shows the PCRprofile expected for the nusA+ allele (lane 7). We have notdetermined whether the chloramphenicol resistance of thisbacterium is due to a spontaneous mutation or an illegitimaterecombination event that transferred the cam' cassette toanother position on the bacterial genome.

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FIG. 4. Determination of nusA allele by PCR analysis. Twoprimers that match the DNA sequences located within the nusAcoding region were used in the PCRs. DNA from the following strains(relevant genetic markers are listed in brackets) served as templates:K37 [nusA+ rho+] (lane 1), K7314 [nusA+ rho(E134D)] (lane 2),K7314 with a AcI857AnusA533::cam prophage [nusA+ rho(E134D)and AnusA533::cam] (lane 3), K7619-21 [AnusA533::cam rho(E134D)] (lanes 4-6), and K7623 [nusA+ rho+ and a camr marker atan unknown position] (lane 7). DNA size markers, fragments gen-erated from a BstEll digest of A DNA, with relevant sizes listed onthe left, are displayed in lane M. The PCR using these primers shouldyield a 0.9-kb DNA product with a DNA template having a nusA+allele and a 1.7-kb DNA product with a DNA template having aAnusA533::cam allele.

Levels of NusA Protein in the AnusA533::cam rho(E)34D)Strain. As shown by Western blots in Fig. 3B, strains K7619and K7622, having only the AnusA533::cam allele, failed toexpress any detectable levels ofNusA protein (lanes 3 and 4),while strains K7314 and K7623, with the nusA+ allele, asexpected expressed significant levels ofNusA (lanes 1 and 2).Note that K7623 is the chloramphenicol-resistant bacteriumacquired in the selection for AnusAS33::cam mutants in thepresence of the rho+ allele. Probing of the blot with ananti-Rho antiserum showed that extracts from the three rho(E)34D) derivatives (lanes 1, 3, and 4) had roughly equivalentlevels of Rho protein. Presumably, because of autogenousregulation (40), extracts from the rho+ host (lane 2) had aslightly lower level of Rho.

Effect of the AnusA533::cam Mutation on Bacterial Growth.Although the AnusA533::cam rho(E134D) strain K7619grows in a rich medium (LB broth) at both high (420C) and low(320() temperatures, the growth rate in this medium isreduced compared to the growth rate of the nusA+rho(E134D) isogenic strain K7314. The doubling time in LBbroth at 320C is 120 min for K7619 compared to 40 min forK7314. In poor media, such as TB (tryptone broth) andminimal M9-glucose, K7619 fails to grow at 420(. A plasmid,pNAG15, with an insert containing an expressible nusA genecloned independently of the downstream genes (24), restoresnormal growth to K7619. Thus, the poor growth of K7619 islikely to be a direct consequence of the lack ofNusA and nota consequence ofany polar effects on the expression ofgenesdownstream of nusA.

Effect of rhol Mutation. We tested a second rho mutation,rho) (31), for suppressor activity. As in the case ofrho(E)34D), E. coli derivatives containingthe AnusA533::camcassette as the only nusA allele can also be constructed if thebacterium carries the rho) allele (data not shown). Likerho(E134D), rho) suppresses the thermosensitivity of thenusA mutants and the triple nusA) rpsJ-nusE7) rpoA(D305E)mutant K4195 (data not shown). Moreover, like rho),rho(E134D) suppresses the polarity normally imposed by thetrpE9851 mutation (data not shown) (31). Termination activityof rho) was assayed by using the previously describedpKLtR3 vector system. As shown in Fig. 2, rho) reduces

termination at tR3 approximately to the same extent as doesrho(E134D).

Sequencing analysis identified a single base pair change inrho), which results in a substitution ofa glutamic acid codon(GAA) for a lysine codon (AAA) at codon position 352,placing rho) at the C end of the Rho protein. Identical resultshave been obtained by S. Pereria and T. Platt (personalcommunication).

DISCUSSIONThe construction of a bacterium completely lacking in NusAwas made possible by isolation of the rho(E134D) mutation.This mutation, a single nucleotide substitution resulting in achange ofa glutamic acid to an aspartic acid at amino acid 134of the Rho protein, was shown to suppress the thermosen-sitive growth caused by several nusA mutations. Although wewere unable to construct an E. coli with the AnusA533::camallele if the bacterium has a rho+ allele, we were able toconstruct such a strain if it has another rho mutation, rho)(31). An E. coli with the rho) allele, like rho(E)34D), exhibitsan 8- to 9-fold reduction in Rho-dependent termination.Sequence analyses show that rho) is a point mutation chang-ing a glutamic acid to a lysine at amino acid 352 in theC-terminal region, while rho(E134D) changes an amino acidin the N-terminal region of Rho. The fact that rho) andrho(E134D) are located in apparently different regions of rhosuggests that their suppression of nusA mutations is not theresult of a change in a protein-protein interaction but ratherreflects the fact that both mutations cause a similar reductionin transcription termination. However, we cannot rule out thepossibility that these two mutations may affect the samebiochemical activity of Rho.A variety of studies have provided support for the idea of

an interaction between NusA and Rho. However, results ofthese studies are contradictory, with some suggesting thatNusA enhances Rho-dependent termination, while otherssuggest the opposite (11, 15, 16, 18-20, 27, 41-43).

In this paper, we show that NusA, normally essential, isdispensable in E. coli expressing Rho proteins with decreasedtermination activity. This result, indicating that the essentialfunction for NusA in E. coli is to antagonize the effect ofRho-promoted transcription termination, provides a basis forrationalizing the apparent contradictory roles of NusA intranscription elongation. Exaggeration of an RNA polymer-ase pause by NusA has been invoked as a mechanism forensuring the close coupling of transcription and translationessential for regulation at certain bacterial attenuators (re-viewed in ref. 44). Although the termination at attenuators, inmost cases, is Rho independent, this idea provides a frame-work for considering the effect of NusA on Rho action.According to the favored tracking model, Rho binds tomRNA and at the expense ofATP moves toward the pausedpolymerase (reviewed in refs. 11, 12, and 45). It has beenproposed that ribosomes bound to the mRNA can interferewith both the binding of Rho and the progress ofRho towardthe paused polymerase; hence, coupling of transcription andtranslation may also influence Rho-dependent termination(11, 12). Recent studies suggest that movement of the tran-scribing polymerase is discontinuous (2, 4), suggesting anadded reason why factors governing the transcription ratemay be required to ensure proper coupling of transcriptionand translation.We postulate that the essential role of NusA in the bacte-

rium is to ensure tight coupling of transcription and transla-tion (27, 46-48) by prolonging pausing ofpolymerase (15, 16,19) and ultimately reducing Rho-dependent intragenic tran-scription termination. This means that studies of Rho termi-nation in the absence of translation might not be a truereflection of the physiological process. For example, al-

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though the findings of Jin et al. (49) that Rho-dependenttermination is inversely related to the rate of transcriptionelongation appear contrary to our model, it is difficult toassess the physiological significance ofthose studies becausetermination was examined in the absence of translation.Assuming that under conditions favorable for growth pre-

mature termination of transcription is detrimental to thebacterium, NusA could make an essential contribution to cellviability by enhancing pausing of RNA polymerase. Thiswould allow ribosomes to maintain proper spacing with RNApolymerase, facilitating tight coupling of transcription andtranslation that could serve as an impediment to Rho-promoted premature termination. The question then is, ifpremature termination is potentially harmful, why are intra-genic termination signals maintained? Ruteshouser and Rich-ardson (48) proposed that such premature termination, po-larity, serves a physiological role, aborting transcriptionwhen translation is inefficient. We propose that NusA be-comes dispensable under conditions of weakened Rho ter-mination activity (e.g., in the presence of the rhol or E134Dmutations), because Rho-imposed premature termination oftranscription is reduced to a tolerable level.A comparison of two in vitro transcription studies on lacZ

supports this model. Ruteshauser and Richardson (48), usingan in vitro transcription system, found no effect of NusA onRho termination activity at sites within the gene. Kung et al.(14) also observed little effect of NusA on Rho-mediatedreduction in lacZ expression in an in vitro transcriptionsystem. However, the latter workers did find that whentranscription was coupled in vitro with translation, the addi-tion of NusA significantly reversed the Rho-imposed reduc-tion in lacZ expression. Hence, the reduction in Rho-dependent termination within the lacZ gene mediated byNusA depends on a coupling of transcription and translation.Our model of NusA action appears to contradict the

numerous experiments indicating that rather than reducingtranscription termination, NusA enhances it. However, thiscontradiction could be more apparent than real. If the pri-mary role ofNusA is to enhance pausing of the transcriptioncomplex, then the outcome of the pause could be verydifferent depending on where it occurs. Pausing within thereading frame could, as we postulate, facilitate coupling oftranscription and translation and thus block termination.Pausing at the end of a transcription unit, where the RNA isnot translated, could facilitate the access ofRho to the pausedtranscription complex, leading to termination.

It has been proposed that NusA be considered a subunit ofRNA polymerase (28). The fact that E. coli variants cansurvive without NusA, albeit in the presence of a secondmutation, demonstrates conclusively that productive in vivotranscription occurs without NusA. By this strict definition,it can be argued that it might be better to consider NusA anaccessory factor rather than a subunit.

The authors thank S. Adhya and D. Bear for anti-Rho antiserum,S. Kushner for plasmids, and Y. Nakamura for nusA mutants. D.Court, V. DiRita, M. Cashel, and J. Richardson are thanked forhelpful discussions and bacterial strains. V. DiRitaandJ. Richardsonare thanked for critical reading of the manuscript. This work wassupported by Public Health Service Grants A111459-10 and M01-RR00042 for sequence analysis.

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