J. Mol. Biol. (1977) 112, 423-436

Transcription Termination at the End of the Tryptophan Operon of Escherichia coli LEONARD P. GUARENTE1, DAVID H. MITCHELL2 AND JON BECKWITH1

iDepartment of Microbiology and Molecular Genetics Harvard Medical School Boston, Mass. 02115, U.S.A. and 2Department of Cell Physiology Boston Biomedical Research Institute Boston, Mass. 02114, U.S.A. (Received 28 September 1976, and in revised form 10 January 1977) The nature of transcription termination at the end of the trpt operon of Escherichia coli has been partially characterized. Strains were constructed in which the lac genes were transposed to a chromosomal site near the trp operon. When the lac promoter is deleted from such strains, lac expression depends upon readthrough from the trp operon and can be used to detect mutations which alter normal termination of transcription of the ~rp operon. I t was possible to isolate mutations in the region where transcription of the trp operon normally terminates, thereby allowing substantial readthrough into the lac operon. In addition, a mutation which mapped at or near the rho locus was isolated which allowed efficient readthrough from the trp operon into the lac operon, across a region of about 1800 bases beyond the terminus of the ~p operon. These results suggest that rho factor is involved in normal termination of transcription of the trp operon. 1. I n t r o d u c t i o n The molecular basis of transcription termination has been studied in several systems

in Escherichia coll. A bacterial protein, rho protein, will promote termination of transcription in vitro at specific sites on phage 2 D N A (Roberts, 1969). Termination at these sites in vivo is presumed to prevent transcription of genes essential for A growth. The N protein of ~ is thought to overcome transcription termination b y rho protein at these sites, and thus permit ~ growth (Roberts, 1969 ; Franl~]in & Yanofsky, 1976). The requirement for N protein for ~ growth can be overcome b y mutants defective in rho protein (Reyes et al., 1976; Das et al., 1976; Inoko & Imai, 1976). These findings strongly suggest the involvement of rho protein in ~ messenger R N A termination

in vivo. Transcription termination has also been implicated in the effects of polar mutations in bacterial operons. Polar mutations, including nonsense, frameshift, and insertion mutations in one gene of an operon, result in reduced expression of genes promoter~fAbbreviations used (Taylor & Trotter, 1972) : ara, bgl, gal and tacPOZYA are operons for genes involved in the catabolism of arabinose, fl-glucosides, galaetose and lactose, respectively. ilv and trpEDCBA are operons for genes involved in the biosynthesis of isoleucine-valine and tryptophan, lacI and trpR are control genes for the lac and Srp operons. XG, 5-bromo-4-chloro3-indolyl-fl-n-galactoside. 28

423

424

L. P. G U A R E N T E , D. H. M I T C H E L L AND J. B E C K W I T H

distal to the mutational site (Newton e~ al., 1965). Certain mutations which suppress these polar effects have been shown to alter rho protein (Kern & Yanofsky, 19765; Richardson et al., 1975). Transcription termination has also been found to play a role in the regulation of the trp~ operon of E. c~oli. A certain percentage of transcripts initiated at the trp operon promoter terminate at a site 30 to 60 nucleotide pairs before the first structural gene of the operon. This site is called the trp attenuator (Bertrand et al., 1975). The extent of termination is greatest when cells are growing in the presence of excess tryptophan. Termination of transcription at the trp attenuator can also be observed in a purified transcription system, in the absence of accessory factors including rho (Lee e~ al., 1976). I n rive, certain the mutations have been shown to exert a small effect upon termination of trlv m R N A at the attenuator (Kern & ¥anofsky, 1976a). So far, there has been little information on the nature of transcription termination signals at the end of bacterial operons. I n vitro experiments have shown t h a t rho protein can cause termination of transcription at or near the end of the gal operon (deCrombrugge et al., 1973), but there is no evidence to support a role for rho protein in this system in rive. Transcription termination signals at the end of bacterial operons are difficult to analyze genetically, unless they play some other regulatory role, as is the case with the phage ~, and trio attenuator termination signals. While mutations altering prometer sites can have profound effects on opcron expression, mutations affecting transcription terminators for operons are not expected to change the normal functioning of t h a t operon. One approach in overcoming this problem is to analyze the effect of termination signal mutations on transcriptional readthrough into neighboring operons. Such an analysis has been initiated in a s t u d y of fusions of t h e / a c operon to the trp operon (Mitchell e~ al., 1975,1976). In these studies, the lac operon has been transposed to a site close to the trp operon, with the tomB locus in between the two operons (Fig. 1). When tonB mutants are selected, some are deletions fusing the ~ c genes to the trlV controlling elements. Such deletions must delete any termination signal t h a t exists at the end of the trp operon (t~rp, Fig. 1). Some of the deletions which generate fusions leave the last structural gene of the trl~ operon, trpA, intact (class I, Fig. 1). This indicates t h a t ttr p must lie beyond the terminus of tr1~A (Mitchell et al., 1976). I n order to establish a system for characterizing t~rp, we have isolated a series of tonB deletion strains in which the b~c operon is brought close to, b u t not fused to the trp operon (class II, Fig. 1). In these strains, t~rp is intact, but t h e / a c promoter is at least partially deleted. Since the strains are thus ~ c - , mutations which alter t~rp, or its functioning, will result in fusion of/~c to the trip operon, and a ~o + phenotype. The nature of ttr p can now be analyzed b y selecting for ~o + revertants in such strains~

2. Materials and M e t h o d s (a) Chemicals, media and c~say8 5-Bromo-4-chloro-3-indolyl-~-D-galactoside (XG) is a non-inducing substrate of flgalactosidase, which is hydrolysed to release modified indolyl moieties that dimerize to produce the blue dye indigo. On minimal agar containing XG, colonies which have high levels of ~-galactosidase are deep blue, while colonies with low, or no levels are pale blue, or white. t See footnote on page 423.

TRANSCRIPTION

TERMINATION

OF T H E t ~ O P E R O N

425

M63 minimal m e d i u m (Pardee et al., 1959) was used for minimal agar, a n d phosphate buffer m e d i u m (Reznikoff et al., 1974) was used to grow cells for assays. Media used to grow cells f o r transaeetylase assays routinely were supplied with 20 gg t r y p t o p h a n / m l a n d 0"2~/o glucose. Isopropyl-~-D-thiogalactoside a t a final concentration of 10 - a M was a d d e d to m e d i a for induction of the lac operon, t o n B - strains were supplemented with 0.006 M-sodium citrate when grown on minimal agar. ~-galactosidase a n d transacetylase assays were performed as described b y Miller (1972). The error in the transacetylase assay was ± 15~o. T r y p t o p h a n synthetase A was assayed as described b y Smith & Yanofsky (1962), except 0.8 gmol r a t h e r t h a n 0.4 gmol indole was used in the reaction mixture. All genetic techniques, including P1 transductions, bacterial matings and plate lysates, were performed according to Miller (1972). tonB selections were performed as previously described (Gottesman & Beckwith, 1969). (b) Bacterial s~rains and strai~ co~truction Bacterial strains used are listed in Table 1. G1 to G5 are AtonB strains derived from X7800. X8605, Ai126 a n d Ai232 are AtonB strains derived from X7700 (Miller ct al., 1968; Schmeissner et al., 1976). The t~pR- m u t a t i o n (Imamoto et aL, 1966) was introduced into X8605, Ai126, a n d Ai232 b y m a t i n g with D7011.1 a n d selecting val r, s t r A recombinants. The val r m a r k e r used lies close to t r p R - . Recombinants were screened for 5-methyl t r y p t o p h a n resistance, which indicates the presence of the t~ivR- allele (Morse & Yanofsky, 1969). F36a, F20 a n d XW211 are trp--lac fusion strains (Mitchell et al., 1975). TABLE 1 Bacterial strains

CA150 CA152 CA7092.1 ]:)7011.1 X7700 X7711 X7800 X7811.1

Hfr Hfr Hfr Hfr

Hayes/acI3,Z2 Hayes lacZ Ul18 Hayes ara val r (lac-pro)Xlll Cavalli trpR val r F - ara J(lac-pro) X l l l , strA ~80d lac + F - ara A(lac-pro)XIII zJ(trpCBA-tonB-lacZ) 484 strA F - trpR A(lac-pro) X l l l strA ~b80d lac + F - trpR ~lac U169 Atrpl415 strA

All strains are from the collection of Beckwith. The t r p R + m a r k e r was introduced into strains carrying the t r p R - allele b y m a t i n g with CA7092.1 a n d selecting val r, s t r A recombinants, or screening for a r a - , strA recombinants on arabinose MacConkey agar containing s t r e p t o m y c i n if the recipient a l r e a d y carried the val r mutations. R e c o m b i n a n t s were tested for 5-methyl t r y p t o p h a n sensitivity. (c) Construction of t o n B deletion strains car~]i~4 a deletion of the trp attenuator Deletion 1415, which removes t h e top a t t e n u a t o r a n d extends into trpC (Bertrand & Yanofsky, 1976; Fig. 1), was introduced into a t r p R - , pro + derivative of X7711 (Miller ctal., 1970) b y P1 transduction, selecting for a b i l i t y to utilize indole as source of t r y p t o phan. Indole utilization is dependent u p o n expression of the trpB gene, which is deleted in X7711. The t r a n s d u c t a n t s were also tonB + . This strain was n a m e d X7811.1. The tonB deletion X8605 was introduced i n c/s to deletion 1415 b y P1 transduction, selecting for tonB, a n d screening for blue color on X G agar, a n d the t~p- phenotype. The tsul, Ivsu2 a n d p s u 3 m u t a t i o n s were introduced into this strain b y the technique described below. (d) Construction o] strains containing t s u l , psu2 and psu3 I n order to s t u d y t h e effect of t s u l in a strain b a c k g r o u n d which h a d n o t been m u t a genized, we have introduced tsul, along with the psu2 a n d psu3 mutations, into strain X7811.1. F i r s t X7811.1 was m a d e i l v - b y P 1 cotransduction with bgl + (Prasad & Schacfler,

426

L.P.

G U A R E N T E , D. H. M I T C H E L L AND J . B E C K W I T H

1974). The suppressor mutations t ~ l , psu2 a n d p ~ 3 were then introduced into these strains b y P1 cotransduction with ilv +. The t r a n s d u c t a n t s were scored for their ability to suppress the lacZ2 polar mutation. This was done b y crossing the potential polarity suppressor strains with an Hfr strain carrying/acZ2 (CA150) a n d selecting recombinants on-melibiose minimal agar with streptomycin at 42°C. U n d e r these conditions only recombinants in which the galactoside permease gene is expressed, due to suppression of polarity, will grow. The class I I tonB deletions were then introduced b y P1 transduction into strains carrying the suppressors b y selecting for trp +, a n d screening for blue color on X G agar. t~ul, psu2 and psu3 were also introduced into X8605, F36a, F20, XW211 a n d CA152 a n d tsul was introduced into the ~ r p R - E - A - derivative of W3110 (Korn & Yanofsky, 1976a,b) b y P1 cotransduction with ilv +. Transductants were scored as described above. Mating with CA150 can also be used to indicate suppression of polarity in strains conraining trp-lac fusions. This is done b y omitting citrate from the agar, which selected for the tonB + character, tonB + recombinants must lose the trp-lac fusion, so a melibiose + phenotype m u s t come from suppression of polarity of the/acZ2 mutation. (e) ~election of class I I t o n B deletion strains Method 1 Cells were treated with the phage a n d colicin preparation used to select ~onB m u t a n t s (Gottesman & Beckwith, I969), and grown in broth for 3 h to allow lysis of sensitive cells. The survivors were pelleted b y centrifugation, resuspended in broth, and a portion spread on minimal glucose X G agar to yield about 103 colonies/plate. This method eliminated the fi-galactesidase released from lysed cells, thus avoiding a high background of blue color in the agar. Blue colonies producing fi-galactosidase constitutively, appeared at a frequency of 0"5~/o. They were picked, purified on minimal X G agar, a n d then screened on lactose tetrazolium agar for their lac character. Colonies which were red on lactose tetrazolium agar (lac-) were presumed to contain ~onB deletions of the class sought (class II). They were found at a frequency of 0-006% among tonB mutations. Method 2 Cells were treated with the agents used to select tonB m u t a n t s , and spread directly on lactose tetrazolium agar. lac- colonies appeared at a frequency of 5%. A b o u t 16 laccolonies/plate were picked, purified, a n d tested on minimal X G agar. Colonies which were blue on this medium were presumed to be of the class sought. They were found at a frequency of 0"002% among tonB mutations. (f) Determining the exten$ of the class I I t o n B deletions The DNA which includes the tonB deletions was incorporated onto phage ;t189 {Fig. 2) b y the following technique. Plate lysates of this phage on the class I I , tonB deletion strains were made. Portions of the lysates were t h e n mi~ed with a / a c deletion strain, a n d spread on X G agar. I n the case of each £tonB strain, phages which made blue plaques were found with a frequency of 10 -3 to 2 × 10 -~. This consistently high frequency of phages t h a t incorporated a functional lacZ gene indicates t h a t they arose b y homologous recombination between /~189 a n d the £tonB strain to incorporate the ~rpB-A-AtonB-lacZ region of the bacterial chromosome onto the phage chromosome. W h e n ~189 was grown on the $onB + parent strain, no /acZ phages were generated. Presumably, this is because the a m o u n t of DI~A between the ~rp a n d / a c operons is too large to be packaged in the ~ phage particle. I n addition to the class I I tonB deletions, the $rp--lac fusion W227 (Fig. 3), which is k n o w n to remove the terminus of the trpA gene, was incorporated into ~189. ~W227 Z + was grown on a strain with a lacZ amber m u t a t i o n , a n d a phage which incorporated the lacZ m u t a t i o n was isolated. On X G agar, this phage made white plaques on a lac- strain, a n d blue plaques on a lac- S u + amber strain. Phages A zlW227Zam a n d each of the Aclass I I ~tonB Z + phages were cosedimented in a CsC1 gradient in a type 41 fixed-angle rotor at 35,000 revs/min for 22 h. The gradients were fractionated into 200 to 250 2-drop fractions, a n d the fractions were titrated b y

T R A N S C R I P T I O N T E R M I N A T I O N OF T H E trp O P E R O N

427

observing phage plaques on a lac deletion strain on X G agar. The difference i n the peak fractions of X AW227 Zam a n d )~ class I I ~tonB, Z + represents the difference i n density of the phages due to their difference in £)NA content (Weigle et al., 1959). tonB deletion W227 a n d all of the class I I tonB deletions have one end within the lac promoter operator region. Since the entire length of this region is only 122 base-pairs (Diekson eta/., 1975), a n y major difference in density between the phages carrying these deletions must be due

to variations in endpoints between the tonB and trp loci. The gradient was checked by determining the density difference between ~W227 lacZ + and ~W227 /acZW4680, which carries the 1300-base-pair internal lacZ deletion W4680 (Malamy et at., 1972).

3. Resulta (a) Gonstruction of t o n B deletion strains We have isolated strains carrying tonB deletions with one end in t h e / a c p r o m o t e r operator region, and the other in or beyond the tonB locus, but leaving the trp operon intact (Fig. 1, class II). These were isolated from strain X7800, which contains a trp P

I

lac tonB at/80 I P O Z Y A at/80 [ I [ I11 1 I I i

E D C 8 A l,,p

I

i

I

'

1415

I

~

', ,[

I

[ ............

clossI _class H

FI(~. 1. Structure of trp-tonB-q~80d lac region of X7800 chromosome, tonB deletions that remove tL,~ (class I) can result in fusion of the lae operon to the trp controlling elements.

deletion of the n o r m a l / a e chromosomal site, and a transposition of lac to the ~80 attachment site. Two approaches were used to detect strains carrying class I I deletions. First, tonB mutants of X7800 were selected on minimal glucose agar containing XG, but no tryptophan, in order to retain the trp structural genes intact. Deletions which extend into t h e / a c I gene, or delete part but not all of/acP, can result in a constitutive synthesis of fl-galactosidase higher than the parent strain. Such strains will appear as darker blue colonies on X G medium. The lacP (lac-) are distinguished from t h e / a c I (/ac +) deletions b y purifying and testing for t h e / a c character on lactose tetrazolium a g a r . / a c - strains carrying the tonB deletons G3, G4 and G5 were isolated in this manner. The second method involved selecting tonB mutants of X7800 on lactose tetrazolium agar./a6- colonies were picked, purified, and their trp character tested on X G minimal glucose agar without tryptophan, Strains which formed blue colonies on this medium were presumed to contain tonB deletions which left intact the structural genes of the /ac operon and had residual promoter activity. The remaining /acdeletions either removed most of t h e / a c promoter, or extended into t h e / a v structural genes, tonB deletions G1 and G2 were isolated in this manner. Also used in this study were class I I tonB deletions X8605, i126 and i232, which were isolated b y a technique similar to our first method (Miller et al., 1968; Schmeissner et al., 1976). (b) Absence of readthrough from the trp operon into the lac operon in class I I t o n B deletion strains We have examined the residual/ac expression in the class I I tonB deletion strains to determine f l i t results from readthrough from the trp operon, fl-Galactosidase levels of each ,~tonB strain were determined in trpR- and trpR + backgrounds in the presence of exogenous tryptophan. In trp-lac fusion strains, the same trpR- mutation

428

L . P . G U A R E N T E , D. H. M I T C H E L L AND J. B E C K W I T H

has previously been shown to result in as much as a 15-fold increase in lao operon expression (Mitchell etal., 1975). However, no effect of the t r p R - allele on lao expression was seen in a n y of the class I I tonB deletion strains except X8605, where a twofold derepression occurred (Table 4). When the color reaction on lactose MacConkey indicator agar was used to monitor lac expression, again only X8605 showed an effect of the trpR- mutation. I t has previously been found in some trp-lac fusion strains t h a t the amount of readthrough from the trp operon into the lac operon is underestimated when ~galactosidase levels are used as a measure of this readthrough. When thiogalactoside transaeetylase (product of the lacA gene) levels or lac m R N A is assayed in these strains, a higher degree of readtlirough is observed (Reznikoff etal., 1974). To determine if X8605 is a fusion of this kind, thiogalactoside transacetylase assays were performed. I n both the trpR + and trpR- backgrounds extremely low levels of the enzyme were observed, indicating t h a t readthrough from trp into lac in X8065 is very slight, as compared with t h a t in trp--lac fusion strains previously studied. We therefore infer t h a t the region where trp transcription normally terminates is left intact in all of our class I I tonB deletion strains. (c) Mapping the extent of the class I I t o n B deletions We wished to determine the distance from the terminus of the trp operon (the end of the trpA gene) to the beginning of the lac operon in each of the class I I tonB deletions. This was done by recombining each deletion into the genome of a Atrp-lac transducing phage (see Fig. 2, and Materials and Methods) and comparing the density of each deletion with one deletion (W227) of known extent, which ends late in trpA /ac trp y z 189 A 8 C"

A

N

cI

R

k trp-loc 189 FIG. 2. Structure of Atrp-lac 189 phage (Barnes etal., 1974). This phage carries intact the trpA and trpB genes, and the lacy gene, but is partially deleted for the trpC and lacZ genes. TABLE

2

Mapping of class I I t o n B deletions tonB deletion strain

Approximate distance in base-pairs of tonB deletion joint from the terminus of the trpA gene

F36A G5 X8605 G1 G2 G3 i126 i232 G4

0 1400 1800 2300 2700 5500 5500 6400 6700

tonB deletions were mapped as described in Materials and Methods. As controls, gradients were run in which phages differed only by an internal deletion (W4680) or an insertion (MS319) in lacZ whose sizes had been previously precisely determined (Malamy etal., 1972). The determination of the extent of W4680 or MS319 by our technique agreed closely with that of Malamy s~al. The error in the determination of the position of peaks on the gradient could result in an error for each deletion of approximately ± 500 base-pairs.

T R A N S C R I P T I O N T E R M I N A T I O N OF T H E trp O P E R O N

429

lrp

P I

E

I

I

O

I

C

I

BA

I

)'on B a l l 80

t

I

I

~/ m

I

t

I

I PO z Y A olt 80 'III I' ', ', ',

G4 i 252

,

i l26~ G5 G2 GI X8605

I

I

.. ....

, ._

G5

l

W227

,i

4 8 4 (stroin X7711)

Fro. 3. Class I I tonB deletions, lac endpoints are somewhere in the promoter-operator region, and trp endpoints are described in Table 2. W227 is a trp-lac fusion whose Zac endpoint is in lacP. W227 has an altered trpA (Mitchell st aL, 1976) but can recombine with trpA96 to give trp ÷. trpA96 maps 75 nucleotides from the operator distal end oftrpA (Yanofsky & Horn, 1972). Thus, W227 cannot extend more than 75 nucleotides into the trpA gene. The tonB deletion 484, present in strain X7711, cuts into lacZ on one end, and into trpC on the other. and in lacP. The results of these experiments are presented in Table 2 and in Figure 3. (d) Isolation of transcription termination mutants The strain which has proved m o s t useful, so far, for the isolation of m u t a n t s which abolish termination of trp operon transcription at the end of the operon is ](8605. The t r T R - m u t a t i o n was introduced into X8605 in order to maximize the chances of detecting m u t a n t s which cause readthrough from trp into/ac. This strain grows v e r y slowly on minimal lactose agar, and appears slightly lac + on lactose MaeConkey agar. After ultraviolet light mutagenesis, portions of a culture of X8605 were spread on minimal lactose agar at 30°C. At a frequency of 10-4, colonies larger t h a n the background appeared. These were picked, and purified directly on lactose lYlacConkey agar. The strains t h a t appeared to metabolize lactose more efficiently t h a n the parent were studied further. Three m u t a n t strains were isolated in this manner and were shown to exhibit trTR control of lac expression. We presume t h a t this class of m u t a n t s could arise b y one of two types of events: (1) a deletion or point m u t a t i o n of the termination signal beyond the end of the trT operon, or (2) a m u t a t i o n which alters some component of the transcriptional apparatus, allowing transcription to proceed beyond this signal, l~utations of t y p e (2), in all likelihood, would be genetically unlinked to the tonB deletion. I n order to determine whether a n y of the three mutations was unlinked to the tonB deletion, P1 transduetion analysis was performed. W e began with a strain containing a large tonB deletion, which deleted p a r t of trT, and p a r t o f / a c (X7711, Fig. 3), and which also contained the t r p R - mutation. This strain was transduced to trp + b y P1 stocks which h a d been grown on each of the three m u t a n t strains. The trp + transductants were t h e n screened f o r / a c phenotype. P1 lysates of two of the three m u t a n t strains yielded trT + transduetants which w e r e / a c +. The high efficiency of readthrough from trp into lac in these two strains, RT17 and TR38 (Table 5), and their linkage to the trp locus strongly suggest t h a t these mutations result in a defective trp termination signal (type (1) mutations). A P1 lysate of the third m u t a n t yielded trp + transductants which w e r e / a t - , suggesting t h a t this m u t a n t is of the second t y p e discussed above. Experiments, described below, verify t h a t a suppressor of trp operon transcription termination, genetically unlinked to the trp locus, is the cause of the

430

L.P.

GUARENTE,

D. H. M I T C H E L L A N D J . B E C K W I T H

/ac + c h a r a c t e r o f t h i s m u t a n t . This m u t a n t s t r a i n , X8605 tsul ( t e r m i n a t i o n s u p p r e s s o r 1); is t h e m a i n s u b j e c t o f t h i s s t u d y . (e) M a p p i n g of the t s u l mutation tsul was f o u n d t o be 9 0 % e o n t r a n s d u e i b l e b y P1 w i t h t h e ilv locus. T h r e e - f a c t o r crosses g a v e t h e o r d e r bgl.ilv.tsul. Since, as we show below, tsul suppresses p o l a r i t y , a n d k n o w n rho m u t a t i o n s which suppress p o l a r i t y a r e g e n e t i c a l l y l i n k e d t o t h e ilv locus in t h e s a m e o r d e r (]:)as et al., 1976; I n o k o & I m a i , 1976), i t s e e m e d possible t h a t t s u l was a rho m u t a t i o n . I n o r d e r t o t e s t t h i s p o s s i b i l i t y , we h a v e d e t e r m i n e d t h e l i n k a g e o f tsul t o psu3, a p o l a r i t y s u p p r e s s o r also c o t r a n s d u c e d b y P1 w i t h ilv. B o t h r e c o m b i n a t i o n a n d c o m p l e m e n t a t i o n d a t a i n d i c a t e t h a t psu3 is in t h e s a m e gene as o t h e r rho m u t a t i o n s ( K o r n & Y a n o f s k y , 1976b; Y a n o f s k y , p e r s o n a l c o m m u n i c a t i o n ) . W e h a v e f o u n d t h a t t h e r e c o m b i n a t i o n f r e q u e n c y b e t w e e n t s u l a n d psu3 is ~ 1 % (Table 3).

TABLE 3 t s u l maps at the r h o / o c u s

Donor

Recipient

Number of ilv + transductants

Number of ilv + transductants which were rho +

ilv +, psu3 ilv +, rho + ilv +, tsul ilv + rho +

ilv-, tnul ilv-, tsul ilv- psu3 ilv- psu3

175 52 100 50

0 47 0 42

P1 stocks which had been grown on the donor strains were used to transduce the recipient strains to ilv +. The itv- taul recipient was in the strain background of X7800. I t contained a lac operon with the strongly polar lacZ mutation Ul18. If this strain contains the rho + locus, it fails to grow on melibiose at 42°C due to polarity, t~ul and psu3 suppress lac Z U l l 8 polarity, and allow the strain to grow on melibiose at 42°C. The ilv- psu3 recipient strain was in the background of CA152. In this background, both psu3 and tsul cause the strain to be temperature-sensitive for growth on minimal glucose agar, growing at 37°C but not at 42°C. The transductants which were not temperature-sensitive for growth were presumed to contain a rho + locus. (f) t s u l suppresses t r p termination O u r d a t a i n d i c a t e t h a t lac expression in X8605 tsul is a r e s u l t o f r e a d t h r o u g h f r o m t h e trp o p e r o n (Table 4). I n o r d e r to f u l l y assess t h e e x t e n t o f r e a d t h r o u g h f r o m trp in X8605 tsul, t h i o g a l a c t o s i d e t r a n s a c e t y l a s e a s s a y s were p e r f o r m e d . T h e c o n t r o l s t r a i n in t h e e x p e r i m e n t s , X W 2 1 1 , carries t h e m o s t efficient trp-lac fusion i s o l a t e d t h u s far (Mitchell et al., 1975). W h i l e t h e psu3 m u t a t i o n r e p o r t e d b y K o r n & Y a n o f s k y allows some r e a d t h r o u g h from trp i n t o lac, tsul allows v e r y efficient r e a d t h r o u g h in X8605 (Table 5). To d e t e r m i n e i f a n y o f t h e r e a d t h r o u g h in X8605 conferred b y t h e suppressors is a r e s u l t of d e c r e a s e d t e r m i n a t i o n a t t h e trp a t t e n u a t o r site, t r y p t o p h a n s y n t h e t a s e A levels were d e t e r m i n e d , tsul h a d no effect on t r y p t o p h a n s y n t h e t a s e A levels, a n d in this s t r a i n b a c k g r o u n d psu2 a n d psu3 also s h o w e d no effect (Table 6). I n a d d i t i o n , a d e l e t i o n o f t h e trp a t t e n u a t o r in cis to AtonB X8605 r a i s e d t h e thiog a l a c t o s i d e t r a n s a c e t y l a s e levels o f all s t r a i n s t e s t e d w i t h o u t affecting t h e r a t i o o f a c e t y l a s e levels o f a s t r a i n w i t h tsul, t o a n isogenic s t r a i n w i t h a w i l d - t y p e rho locus. I n a d d i t i o n to d e m o n s t r a t i n g t h a t t~ul does n o t affect t e r m i n a t i o n a t t h e trp a t t e n u a t o r in this strain, t h i s e x p e r i m e n t shows d i r e c t l y t h a t r e a d t h r o u g h i n t o / a c conferred

TRANSCRIPTION

TERMINATION

O F T H E trp O P E R O N

431

TABLE 4

Levels of fl-galactosidase in X8605 and X8605 t s u l in a t r p R + and t r p R - background ~-Galactosidase units¢ X8605 trpR + X8605 t r p R X8605 tsul trpR + X8605 tsul trpRX8605 tsul 1415 trpRXW211 trpR-

15 35 40 300 550 3000

1415 is an internal deletion of the trp operon which removes the a t t e n u a t o r site. •1415 and tonB deletion X8605 were constructed in cis as described in Materials and Methods. Cultures were grown, and fi-galactosidase was assayed as described in Materials and Methods, fLGalactosidase units expressed according to Miller (1972).

TABLE 5

Transacety~ase levds in X8605 and X8605 lac + mutants Strains

X8605 trpRX8605 RT17 trpRX8605 RT17 trpR + X8605 RT38 trpRX8605 RT38 trpR + X8605 psu3 trpRX8605 tsul trpRX8605 1415 trpRX8605 1415 tsul trpRXW211 trpR-

Transacetylase Stimulation of readthrough levels by rho mutations 1.7 68 9.6 37 2-2

11

6-fold 41 -fold

70 3.5 130 100

37-fold

Cultures were grown, and thiogalactoside transaeetylase was assayed as described in Materials and Methods.

TABLE 6 Tryptophan synthetase A protein levels in X8605 and its derivatives T r y p t o p h a n synthetase A protein activity X8605 X8605 X8605 X8605 X8605 X8605 X8605

trpR + trpRpsu2 trpRpsu3 trpRtsul trpRtsul rill23 trpR1415 trpR-

1 24 27 32 25 27 90

Tryp~ophan synthetase A protein was assayed as previously described (Smith & Yanofsky, 1962), except 0.8 pmol of indole was used in the incubation mixture instead of 0.4/zmol.

432

L . P . G U A R E N T E , D. H. M I T C H E L L AND J. B E C K W I T H

by tsul is coming from the trl~ operon, and not another operon under trpR control. These data suggest t h a t tsul suppresses termination of transcription at the end of the trp operon in X8605. (g) Effect of t s u l on readthrough in other class I I t o n B deletions None of the remaining class I I tonB deletion strains shows readthrough from the trp operon into t h e / a c operon. However, when tsul was introduced into these strains, it was observed that those tonB deletions ending closest to the trp operon showed considerably elevated levels of transacetylase (Table 7). The increase in transacetylase in these strains is presumed to come from readthrough from the trp operon, TABLE 7 Effect of t s u l on clazs I I £ t o n B strains Strain

Thiogalactoside transacetylase levelst

G5 tsul trpRtrpR + X8605 tsul trpRtrpR + G1 tsul trpRtrpR + G2 tsul trpRtrpR + G3 tsul trpRi126 tsul trpRi232 tsul trpRG4 tsul trpR-

27 O.5 70 5.9 43 3"4 30 9 7 6 6 0.4

This series of strains was constructed as described in Materials and Methods. The units are expressed as the percentage of XW211 trpR- transaeetylase levels. t The thiogalactoside transaeetylase in the rho + derivatives, which is not under trpR control and presumably is a result of rvsidual activity of/acP, has been subtracted in each strain. since it is under trpR control. The lack of correlation in the amount of readthrough of lac from the trp operon with distance (Table 2) in G5 and X8605 m a y be due to the imprecision of measurements of deletion lengths, tsul had practically no effect on the tonB deletions which left the most D N A between the trp and lac operons. A similar pattern was seen with psu2, but with each tonB deletion strain, the amount of readthrough was less than 10% t h a t conferred b y tsul (unpublished data). (h) Effect of t s u l on polar mutations, and on trp-lac fusions Mutation tsul was introduced b y P1 transduction into two different strain backgrounds, both of which contained a lac operon with the strongly polar lacZ mutation U l l S . I n both backgrounds tsul suppressed the polar effect of Ul18 on lacA to a greater degree than did psu2 or psu3 (Table 8). The effect of tsul and psu2 on trp-lac fusion strains was examined (Table 9). The very efficient fusion W211 was unaffected b y tsul or psu2, demonstrating t h a t the suppressors do not act by relieving transcription termination within the lac operon before la~A. Fusions F20 and F36a are quite inefficient for reasons t h a t are not

TRANSCRIPTION

TERMINATION

O F T H E trp O P E R O N

433

TABLE 8

Ability of t s u l to suppress polarity Strain

Transacetylase levels

CA152 CA152 psu2 CA152 psu3 CA152 tsul

trpR-, E-, A - lacZUll8 trpR-, E -, A - psu3 tacZUll8 trpR-, E-, A - tsul /acZUll8

(0.2 2.2 2.9 14 ~0.2 17 37

:psu2, psu3 and tsul were introduced into CA152, and tsul was introduced into t r p R - , E - , A - , as described in Materials and Methods. trpR-, E - , A - is from the collection of C. Yanofsky. These strains all carry the lacZUll8 polar mutation in a normally located tac region. Transacetylase units are expressed as the percentage of levels in an isogenic lac + control. TABLE 9

Effect of tsul and p s u 2 on t r p - l a c fusion strains Strain F20 F20 psu2 XW211 XW211 psu2 XW211 taul F36a F36a psu2 F36a tsul X8605 1415 X8605 1415 p~u2 X8605 1415 tsul

Transacetylase levels

Stimulation of readthrough by rho mutations

12 48 100 113 119 6.6 61 65 3.5 27 130

4.0 1.1 1.2 9-2 9.9 7.7 37.1

All strains contain the trpR- allele. A1415 has been described (Table 3). X8605 results are presented for comparison. obvious. T r a n s a c e t y l a s e levels in b o t h s t r a i n s a r e c o n s i d e r a b l y e n h a n c e d b y p s u 2 . F 3 6 a efficiency is i m p r o v e d t o t h e s a m e degree b y psu2 a n d b y tsul. I t w o u l d a p p e a r t h a t in t h e s e s t r a i n s t h e fusion j o i n t causes a p o l a r effect on t r a n s c r i p t i o n t h a t is s u p p r e s s i b l e b y s u p p r e s s o r s o f p o l a r i t y ( B e r t r a n d & ¥ a n o f s k y , 1976). (i) Reversal of suppression of t r p transcription termination in X8605 t s u l

by an R N A polymerase mutation We have begun a study of mutations which restore termination at the end of the

trp o p e r o n in t h e p r e s e n c e o f tsul. I n t h e process we h a v e i n t r o d u c e d b y P1 t r a n s d u c t i o n a r f f a m p i c i n - r e s i s t a n t m u t a t i o n o f R N A p o l y m e r a s e , r/f123 ( C h a k a r a b a r t i & Gorini, 1975), i n t o s t r a i n X8605 tsul. T h e level o f t r a n s a c e t y l a s e , w h i c h in X8605 tsul d e r i v e s f r o m r e a d t h r o u g h f r o m t h e trp operon, is r e d u c e d a b o u t e i g h t f o l d b y rill23 (Table 10). T h e rill23 m u t a t i o n does n o t lower t h e levels o f t r y p t o p h a n

434

L. P . G U A R E N T E ,

D. H. M I T C H E L L

AND J. BECKWITH

TABLE 10

Effect of rff123 on trp readthrough in X8605 t s u l Strain

Transacetylase levels

X8605 X8605 tsul X8605 tsul, rill23

1.7 70 8'95

The units are expressed as the percentage of XW211 trpR- transacetylase levels.

synthetase A protein (Table 6). Thus it appears that the mutation restores termination between trp a n d / a c in X8605 tsul. The r/f123 mutation also antagonized the ability of tsul to suppress polarity generated by the/acZ2 polar mutation, as judged b y a mating test (see Materials and Methods). Further experiments to determine the nature of the r/f123 effect are in progress.

4. Discussion We have isolated and characterized a series of deletion strains in which t h e / a c operon is brought close to the trp operon, but in which a barrier to transcriptional readthrough from trp into/ac still exists. Beginning with one of these strains, X8605, we have been able to isolate mutations which to varying degrees eliminate this barrier, and thus put t h e / a c operon under the control of the trp operon regulatory elements. One such mutation, tsul, appears to map in the structural gene for rho protein, a transcription termination factor. While we have no direct evidence t h a t rho protein is altered in this strain, the proximity of tsul to a known rho mutation suggests t h a t the two mutants are alleles of the same gene. In addition, tsul suppresses polarity as do other rho mutations. This suppression occurs w i t h / a c polar mutations and with the apparent polarity observed in trp-lac fusions. The presumed rho mutation allows highly efficient readthrough from trp into/ac, giving approximately 70% of the thiogalactoside transacetylase levels seen in the most efficient trp-lac fusion strains characterized to this time. These findings are consistent with the following hypothesis. There exists a termination signal for transcription beyond the end of the trpA gene (ttTp). This signal must be less than 1400 base-pairs distant from trpA, as the tonB deletion, G5, ends at approximately this distance, and does not remove the barrier to readthrough from trp into/ac. Previous findings suggest t h a t transcription of the trp operon proceeds a distance past the end of the trpA gene that is small compared to trpA itself (Mitchell et al., 1976; Rose & Yanofsky, 1971). Further, since a presumed rho mutation eliminates, to a large extent, the barrier to transcription, it seems likely t h a t rho protein plays a major role in transcription termination at the end of the trp operon. These results provide the first in vivo evidence for a role for rho protein in transcription termination at the end of a bacterial operon. We recognize t h a t there are alternative explanations of our data. For instance, there m a y not be a specific termination signal at the end of the trp operon, but accumulated polarity effects m a y prevent readthrough over long distances. The rho mutation may be so strong as to overcome most of this polarity. However, we feel

T R A N S C R I P T I O N T E R M I N A T I O N OF T H E trp O P E R O N

435

t h a t the postulation of ttrp acted upon b y rho protein is the simplest one, considering our d a t a and those of others. The genetic system described in this p a p e r provides a means of further analyzing ttTp. W e have presented preliminary characterization of two mutations t h a t are genetically linl~ed to the trp region, and which remove the barrier to transcriptional readthrough without affecting the trp operon itself. A determination of the site of these mutations should allow us to locate ttTp relative to the trp operon. This could be done b y analyzing small deletions of this class using either D N A heteroduplex or restriction enzyme analysis. The ability of tsul to suppress trp operon transcription termination, and lacZ2 polarity is reversed b y a rffampiein-resistant m u t a t i o n of R N A polymerase, rill23. Some spontaneous rifampiein.resistant m u t a n t s of strain X 8 6 0 5 - t s u l also reversed the suppressor activity of t s u l . One possible explanation for these results is t h a t the suppressor activities of t s u l result from an impaired interaction between R N A polymerase and rho protein caused b y the t s u l mutation. Certain R N A polymerase mutations, like r i l l 2 3 , m a y restore such an interaction, allowing normal transcription termination and operon polarity to occur. Alternatively, the altered R N A polymerase in the r i l l 2 3 m u t a n t m a y be able to terminate R N A synthesis in the absence of rho in regions which are normally dependent on rho. We thank C. Yanofsky, W. Reznikoff and M. Malamy for bacterial and phage strains. We also thank R. MacGillivray and A. McIntosh for cheerful assistance. This work was supported by a National Institutes of Health predoctoral training grant and by a National Institutes of Health grant (GM13017) to one of us (J. B.).

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