J. Mol. Biol. (1969) 41, 329-339

Re-initiation of Polypeptide Synthesis and Polarity in the Zac Operon of Escherichia coli

AUSTIN NEWTON

Program in Biochemical Sciences

Department of Biology Princeton University

Princeton, N.J. 08540, U.S.A.

(Received 14 October 1968, and in revised form 13 December 1968)

Strains of Escherichia coli which contain two nonsense mutations of either the z and y genes or the z gene of the lac operon have been constructed and examined for levels of transacetylase activity. Comparison of the polarity of these double mutants to the polarity of the parent strains, which contain only one of the mutations, supports the following conclusions. (a) Expression of transaoetylase activity by z- nonsense mutants depends upon translation of the y message. (b) Premature chain termination at nonsense triplets in the part of z closest to y (operator-distal region) is followed by re-initiation of polypeptide synthesis predominately at the start sequence for the y message. (c) Premature chain termination at nonsense triplets which map in the middle and first (operator- proximal) part of z is followed by re-initiation of polypeptide synthesis at sites wit&z the z gene message. This interpretation is supported in part by the finding that nonsense mutants of the middle section of z produce a polypeptide corresponding to the C-terminal end of /3-galactosidase.

The nature of the re-initiation sites in z and their relationship to the polarity of z- nonsense mutants are discussed.

1. Introduction The structural genes of the lac operon of Escherichia co&, x, y and a, code for /?- galactosidase, galactoside permease and thiogalactoside transacetylase (transacety- lase), respectively (Fig. 1; Jacob & Monod, 1961; Ippin, Miller, Scaife & Beckwith, 1968). These genes appear to be expressed by their transcription into a single poly- cistronic messenger RNA which is then translated sequentially from the operator- proximal end (Attardi, Naono, Rouviere, Jacob & Gros, ‘1963; Kiho C% Rich, 1965; Alpers & Tomkins 1965; Fowler & Zabin, 1966). When reading of the messenger RNA is interrupted prematurely by chain termination at a nonsense triplet (UAA, UAG, UGA) in the z cistron, the ‘levels of galactoside permease and transacetylase activity may be drastically reduoed (Newton, Beckwith, Zipser & Brenner, 1965; Sambrook, Fan & Brewer, 1967; Zipser, 1967). One of the factors which strongly influences the extent of this reduction, or polarity, is the position of the nonsense mutation in z: the greater the distance between the point of chain termination and the end of z, the more severe the polar effect (Fig. 1; Newton, 1966; Zipser & New- ton, 1967).

During normal translation of the lac messenger, protein synthesis is punctuated by chain termination at the end of one polypeptide message and re-initiation at the

329

330 A. NEWTON

beginning of another. Results of in vitro complementation experiments suggested, however, that polypeptide synthesis may be re-initiated within the x message after premature chain termination in z. It was found that nonsense mutants of the middle section of z produce significant levels of a peptide corresponding to the C-terminal portion of the /Agalactosidase molecule (Newton, 1966).

This paper reports the results of genetic and biochemical experiments which support the conclusion that protein synthesis is first re-initiated at the start sequence for the y message after chain termination at nonsense codons which map at the operator- distal end of the z gene, but that synthesis is re-initiated ineficiently within the z message after chain termination at nonsense codons which map in the middle section of the gene. The nature of the re-initiation sites in z and their relationship to the

polarity of z - nonsense mutants are discussed.

Map position of mutations 2 131 64 281 239 125 82 559 404 750 200 596366 625 659 90 328 707 515

Transacetylase activities of 014845 12 9 IO 14 18 40 42 35 81) 100 14 27 mutants 49

I I 4680

FIG. 1. Map of Zuc operon showing the map positions and transaoetylase activities for indicated nonsense mutations, and complementation groups in z; U178 maps in the same deletion group as NG200. Letter prefixes for mutations have been omitted. Distances between the mutations do not correspond to mapping frequencies. 4680 is an internal deletion mutation of z. Underlined mutations are of the ochre (UAA) class. Other mutations are of the amber (UAG) class. Mapping and characterization of these mutations have been described previously (Newton et al., 1965).

2. Materials and Methods (a) Bacterial strains

All Zac- nonsense mutants used in this study are I?- derivatives of Hfr Hayes. The mapping and characterization of the mutations have been described previously (Newton et al., 1965).

Strains carrying two nonsense mutations of the lac operon can arise in a heterogenote for lac by reciprocal recombination between an episome which carries one of the desired markers and a chromosome which carries the other (Herman, 1965). In order to facilitate curing of the episome during the selection procedure, an F’lac was used whose replication is impaired at 42”C, but normal at lower temperatures (no. 114 in Jacob, Brenner & Cuzin, 1963).

The heterogenote which had been grown at 37°C was first plated on MacConkey agar (Baltimore Biological Laboratories, no. 01-279). The plates were incubated at 37°C for 72 hr and lac+ recombinants purified at 37°C. These recombinants were next streaked on MaoConkey plates and incubated overnight at 42°C. lac- Segregants were purified from colonies which showed sectoring on the MacConkey plates (due to the loss of the recom- binant, Eat+ episome). Double mutants were identified at a frequency of about 1% among these Zac-, F- strains by their failure to recombine when crossed with Hfr strains carrying one or the other of the parent nonsense mutations. These same strains were shown,

RE-INITIATION AND POLARITY 331

however, to recombine with a third, lac- marker. At least two independent isolates of the double mutant and the parent mutants from each selection were examined for trans- acetylase activity (Tables 1 and 2).

(b) Media, cell growth and preparation. of extracts

Liquid cultures were grown in M63 salts (containing/litre: 126 g K2HP0,, 38 g KHsPO,, 20 g (NH,), SO,, and 5 mg FeS0,.7H,O) supplementedwith0.2% glycerol, 5 mg thiamine/l. and 5 x 1O-4 M-IPTGt. Cultures were incubated at 37°C in 50-ml. Erlenmeyer flasks with vigorous agitation. Two ml. of overnight cultures were inoculated into 10 ml. of fresh medium, the cells allowed to grow to late exponential phase and then harvested by centri- fugation. The cells were resuspended in 2.0 ml. of 0.1 M-potassium phosphate buffer (pH 7.2) and disrupted using a Branson model ST7 sonifier. Cell debris was removed by centrifugation. In order to inactivate deacylase activity, the extracts were heated at 70°C for 10 min; denatured protein was removed by centrifugation.

(c) Transacetylase assay

The assay for transacetylase activity is a modified version of one developed by Fox & Kennedy (personal communication to S. Barbour) which measures transfer of the labeled acetate group from acetyl coenzyme A to IPTG. Since the product of the reaction, acetyl- IPTG, is not adsorbed on Dowex-1 acetate, it can be separated from the radioactive substrate. The reaction mixture contained 15 pmoles potassium phosphate, 0.067 pmole [14C]acetyl-CoA (New England Nuclear) diluted with [12C]acetyl-CoA (PBL Laboratories) to give a specific radioactivity of 1.5 pc/pmole, 15 pmoles EDTA and 10 pmoles IPTG in a total volume of 0.15 ml. at pH 7.2. The reaction was initiated by the addition of the reaction mixture to 0.05 ml. of a heat-treated extract. Dilutions were made with a heat- treated extract of MX74, a strain with a deletion of the entire lac operon. The incubation was terminated after 30 min at 37°C by the addition of 0.8 ml. of a 60% ethanol solution. The entire reaction mixture was then passed over a l-ml. bed volume of Dowex-1 (10% cross-linked and in the acetate form: AGI-X2 from Bio Rad) contained in a Pasteur pipette. The column was washed 4 times with l-ml. vol. of distilled water, and a 0.4-ml. portion of the total eluate of 5 ml. counted in 10 ml. of Buhler’s solution (Buhler, 1962). Units of activity are expressed as mpmoles acetyl-IPTG formed per mm. Protein con- centrations were determined on extracts before heat treatment according to Lowry, Rosebrough, Farr & Randall (1951). Specific activities for transacetylase are expressed as percentages of the activity of the lac+ parent strain, MO. This wild-type strain gives a specific activity of approximately 110 units/mg of protein in the above assay. A curve relating acetyl-IPTG formation to amount of MO extract used in the assay is shown in Fig. 2.

Protein (pg)

FIG. 2. Relationship between amount of a heat-treated extract of MO and mpmoles of acetyl- IPTG formed in the standard transacetylase assay (cf. Materials and Methods).

t Abbreviation used: IPTG, isopropyl-fl-o-thiogalactoside. 23

332 A. NEWTON

(d) /%Galu&osidase assay

The enzyme was assayed according to the procedure of Willson, Perrin, Cohn, Jacob & Monod (1964). A unit of activity is defined as the amount of enzyme producing 1 mpmole of o-nitrophenol/min at 28°C.

3. Results (a) Effect of multiple nonsense mutations on expression of a

Since the amber triplet codes for chain termination (Sarabhai, Stretton, Brenner & Bolle, 1964; Stretton & Brenner, 1965) at the level of translation (Champe & Benzer, 1962 ; Garen & Siddiqi, 1962 ; Brenner & Stretton, 1965), a nonsense mutation in a polycistronic messenger should exert a polar effect only if it is read. Thus, the site of chain re-initiation after a nonsense mutation of x can be located by the effect on polarity when a second, operator-distal nonsense mutation of lac is added. If poly- peptide synthesis is re-initiated between the two nonsense mutations in the same operon, the second mutation will be read and contribute to polarity. If synthesis is not re-initiated in the region bracketed by the two mutations, the polarity of the double mutant should be identical to that of a strain containing the operator-proximal mutation alone. This same approach has been used in a study of polarity in the trp operon of E. coli (Imamoto, Ito & Yanofsky, 1966).

(i) Double mutants of z and y

If the lac messenger is translated sequentially from the operator-proximal end so that the expression of a is dependent upon initiation of protein synthesis at t,he beginning of the y message, then y - nonsense mutations should reduce the level of

TABLE 1

Transacetyhe activities of z-y and z--z double mutants

strain kc- Mutation 1st 2nd

Transacetylase o/0 Transacetylase activity

activity? remaining$

Observed Calculated

(a) z-y Doubles MD16 MD16 MD17 MD18 MD19

YA404 10 NG707 27

YA404+NG707 1.8 18 27 NG328 14

YA404+NG328 1.0 10 14

(b) s-z Doubles MD60 MD61 MD59 MD64 MD62

NG200 17 YA625 35

NG200+YA625 18 105 35 YA515 49

NG200+YA515 17 100 49

t Activities are given as o/0 of the wild-type activity as described in Materials and Methods. $ y, Activity remaining expresses the transacetylase activity of the double mutant as a per-

centage of the activity of the strain carrying the first (operator-proximal) mutation alone. The calculated values are arrived at by assuming that the second (operator-distal) mutation contri- butes the same to polarity when added to another nonsense mutant as it does to the wild-type strain.

RE-INITIATION AND POLARITY 333

transacetylase activity of a z- polar mutant by the same percentage that it reduces the activity of the wild-type strain. In order to test this, strains with a nonsense mutation in both z and y were examined (cf. Fig. 1).

Table l(a) shows the transacetylase aotivity of the parent z- strain and the per- centage of activity remaining when nonsense mutations of y are added. It is seen that both the x - and y - nonsense mutations contribute fully to the polarity of the double mutants. Thus, in x- nonsense mutants, the y message is almost always read; there appears to be little expression of the a gene in these z polar strains which is independent of translation through the y message. Imamoto et al. (1966) have reported similar tlndings about translation of the trp operon.

(ii) Double nonsense mutants of z

In order to test for re-initiation of polypeptide synthesis within the x message itself, strains were constructed which contain two nonsense mutations in z. When mutations distributed throughout the gene (see Fig. 1) are examined in this manner, all of them do not show the same behavior.

The transacetylase activities of strains with nonsense mutations in the segment of z closest to y are not reduced by the addition of a second nonsense mutation. Double z - mutants in this part of the gene show about the same polarity as a strain containing the operator-proximal mutation alone (strains MD59 and MD62, Table l(b)). This is

TABLE 2

Transacetykzse activities of z-z double mutants

Strain Zac- Mutation 1st 2nd

Transaoetylase o/o Transacetylase activity

activity? remainingf. Observed Calculated

(4 MD48 MD26 MD28 MD23 MD25 MD58 MD57 MD56 MD65 MD52 MD51 MD50 MD49 MD47 MD46

NC200 18 NG750 14 NG750+NG200 10.3 74 18 YA404 10.5 YA404+ NG200 7.5 71 18 YA559 9 YA559+NG200 3.5 39 18

X82 12 X82 +NG200 3 25 18 u239 4.6 U239+NG200 0.9 20 18 U281 9 U281+NG200 0.6 6 18 X64 4 X64 +NG200 0.4 10 18

(b) MD52 MD79 MD77 MD69 MD68 MD48 MD53

u239 5 NG125 5

U239+NG125 2 40 5 YA559 10

U239+YA659 1.4 28 10 NG200 18

U239+NG200 1.0 20 18

t and #. See footnotes to Table 1.

334 A. NEWTON

in striking contrast to the result described above for strains in which the z-y punctua- tion is bracketed by the two nonsense mutations (Table l(a) ).

Failure of the second mutation to affect polarity does not preclude the possibility that polypeptide synthesis is re-initiated inefficiently in the 2: message after chain termination at the NG200 site; a low level of re-initiation could not be detected by this method due to the generally high levels of transacetylase activity expressed by mutations in this end of the gene. The result does indicate, however, that after chain termination at nonsense mutations in the operator-distal end of x, polypeptide synthesis is first re-initiated in large part at the beginning of the y message.

When polar mutations in the middle and first sections of z are examined individually by the addition of an operator-distal nonsense mutation (NG200), quite different results are observed. Table 2(a) shows that the transacetylase activities of all z- mutants are reduced to some extent when NG200 is included in the same gene. The data also show that the effect of the second mutation on the expression of a is related to the map position of the two nonsense triplets: the transacetylase activities of strains with mutations which map early in x are reduced by more than 80% by the addition of NG200, while the activities of strains with mutations which map close to NG200 are reduced by only about 20%. Since the transacetylase remaining in the double mutant is presumably determined by the frequency at which NG200 is read, the data, presented graphically in Figure 3, indicate that polypeptide synthesis is re-initiated before the NG200 site more frequently in mutants mapping early in x (e.g. X64 and U239) than it is in those mapping closer to NG200 (e.g. YA404 and NG750).

- 70 8 l-4

-60 !i! L 0

-50 .g

=: 0 -40 2

%

-30 .F .F 0 E aI

-20 ; .e .z Y

-10 2

,P

-0 2 X64 U281 U239 X82 YA559 YA404NG750 NG200

Map position of z-mutations

FIG. 3. Effect of NG200 on transacetylase activity when added to different nonsense mutants of z. Bar graph represents transacetylase activities of single nonsense mutants and, -O--O- represents the percentage of the transacetylase activity remaining in the z nonSense mutant after the addition of NG200 to the strain. Data are taken from Table 2(a).

RE-INITIATION AND POLARITY 335

When the complementary experiment is carried out, that is when different operator- distal nonsense mutations are added individually to a Eat operon carrying an amber triplet which maps early in z, U239, similar results are obtained. The second nonsense mutation is most effective in reducing transacetylase activity when it is furthest from the U239 site (Table 2(b)).

Figure 3 shows that re-initiation may be more efficient in some regions of z than in others. Exact mapping of the restarts in z is complicated, however, by lack of informa- tion about absolute distances between markers and the fact that there are a number of regions within x where double mutants cause a measurable reduction in trans- acetylase activity (Table 2).

(b) Products of re-initiation

If polypeptide synthesis is re-initiated in the z message after premature chain termination at Z- nonsense mutations, then amber mutants of z should produce a portion of the C-terminal end of the P-galactosidase molecule. An assay for such peptides is the complementation test for /3-galactosidase activity. It has been shown that peptide fragments of the C-terminal end of the enzyme can be detected in strains with deletion mutations in the first or middle section of z by complementation with strains carrying mutations in the last (operator-distal) section of z (Ullmann, Jacob & Monod, 1967). Three complementation groups, cc, p and W, have been described on the basis of this functional test (see Fig. 1).

It has already been shown that nonsense mutants of the middle section of z do indeed complement in vitro with a mutant (U178) of the operator-distal portion of z (Newton, 1966). More recent results demonstrate that significant levels of in vivo complementation can be measured when concentrated extracts are prepared from partial diploid strains which carry the nonsense mutation on the chromosome and

TABLE 3

(3-Galactosidase activities from wild-type and complementing strains of E. coli

Straint Transacetylase /%Galactosidase activity of F- aotivitys

strainf (units/ml.)

MO 100 635,000 4680/F’lac-,1 78 100 121,000 NG750/F’Zac-u,,, 14 lL2,ooo X64/F’lao-u,,, 5 9000

T See Fig. 1 for description of mutations. $ See Table 1; activities are for strains which carry no episome. 5 All extracts were adjusted to 20 mg protein/ml.

U178 on an episome. The complementation values obtained for two different nonsense mutants and a deletion mutant, 4680, are compared with the ,&galactosidase activity of the wild-type strain in Table 3. These nonsense mutants will also complement with a deletion mutation which covers only the part of z corresponding to the C-terminal end of ,%galactosidase (Wang, unpublished results).

Although the levels of complementation are relatively high considering the polarity of the strains, no conclusions can be drawn from the levels of activity. The efhciency

336 A. NEWTON

x .e .$ x B O.I- 5 ‘2 :: Lk

0.05 -

I I I I IO 15 20 25 Time at 57’C(mrn)

3

Fro. 4. Stability of complemented and wild-type MO /3-galactosidase preparations to heating at 57’C. The extracts described in Table 3 were diluted in PM2 buffer (Ullmann et al., 1968) to 500 pg of protein/ml. 0.6 ml. of each was heated in a water bath at 57’C, samples withdrawn at the times indicated and assayed for j3-galactosidase. The activities at zero time. were normalized as 1.

of nonsense mutants in complementation varies greatly. It can be said, however, that nonsense mutants of z, e.g. X64 and NG750, do produce a portion of the C- terminal end of the molecule. Also, the product of these nonsense mutations of z does not complement to form the wild-type /3-galactosidase as judged by its sensitivity to heating at 57°C (Fig. 4). This instability of the complemented enzyme and its formation in vitro (Newton, 1966) eliminate recombination as an explanation for all of the activity observed in the diploid strains.

4. Discussion The effect of multiple nonsense mutations in x on polarity (Table 2) and the results

of complementation tests (Table 3) show that a nonsense triplet in the middle of z does not necessarily stop all translation of the x message beyond the point of chain termination. Two explanations for translation past a nonsense triplet are re-initiation of polypeptide synthesis and incomplete chain termination, i.e. the triplet is translated infrequently as an amino acid in the strains used. Incomplete chain termination seems an unattractive alternative because of the extreme polarity of those nonsense mutants which map in the first part of the x gene. The mamer in which multiple mutants influence polarity also argues against the latter possibility. If the U239

RE-INITIATION AND POLARITY 337

triplet were translated infrequently as an amino acid, for example, then the effective- ness of an added nonsense triplet in reducing the transacetylase activity of U239 should depend only upon the polarity of the added mutation; this is not the case (Table 2(b) ). Rather it is seen that the contribution of the second mutation to polarity increases with the genetic distance between it and the first mutation (Table 2 and Fig. 3). These results, which indicate that the second mutation is read more frequently when the two mutations are widely spaced, are consistent with an interpretation involving re-initiation : z contains several sites at which synthesis can be re-initiated, and the probability of including them between any two nonsense mutations increases with the distance between the mutations.

The structure and expression of z are pertinent to a consideration of these re-initiation sites. Chemical analysis (Craven, in Newton, 1966; Brown, Koorajian, Katze $ Zabin, 1966) and complementation studies (Ullmann et al., 1968) of /3-galacto- sidase indicate that the 135,000 molecular weight monomer coded by x is a single polypeptide chain. This latter conclusion is supported by the above finding (Table 2 and Fig. 3) that the sites for re-initiation within z do not uniquely define the com-

plementation groups (Fig. 1) as mapped by Ullmann et al. (1967). Thus, the evidence suggests that the ,Egalactosidase monomer is synthesized as one

chain. A natural conclusion is that the start sequences in z may be “generated” after chain termination by (a) misreading of random codon as signals for re-initiation, or (b) recognition of in-phase AUG codons as N-formylmethionine (cf. Sarabhai $ Brenner, 1967). (Presumably, only initiation at in-phase codons would allow con- tinued translation and expression of transacetylase activity.) The latter explanation (b) is attractive since it is known that p-galactosidase contains 24 unclustered meth- ionine residues (Craven, Steers & Anfinsen, 1965; Steers, Craven & Anfinsen, 1965) and the AUG codon will function to initiate polypeptide synthesis in vitro (Ghosh, Sol1 & Khorana, 1967). The inefficiency of these internal restarts (5 to 10% of the wild-type level at the beginning of y) is reminiscent of the inefficient initiators of translation obtained in rII by mutagenesis (Sarabhai & Brenner, 1967; cf. also Martin, Whitfield, Berkowitz & Voll, 1966).

The foregoing has considered only the re-initiation of translation. It can now be asked whether transcription is also initiated within .z in the nonsense mutants. An initiator sequence for gene expression has been observed in. the trp operon of Salmonella typhimurium (Bauerle & Margolin, 1967), and Imamoto & Ito (1968) have shown that there are internal sites in the trp operon of E. coli for initiation of both transcription and translation. If internal initiators of transcription were present within z, premature chain termination would result in the formation of short messenger fragments cor- responding to the operator-distal portion of the operon. This possibility cannot be eliminated in the hc operon, but it seems unlikely after the finding of Imamoto $ Yanofsky (1967a,b) that the principal messenger fragments formed in polar nonsense mutants of trp represent the operator-proximal end of the trp messenger.

Independent of the exact nature of the initiation sequences within z, they may help to explain the position-dependent effect of z nonsense mutations on y and a expression. It seems probable that the “gradient of polarity” (Fig. 1) is composed of at least two components: the flat, rather irregular portion described by nonsense mutations in the middle of x reflects inefficient re-initiation at start signals within the z message, and the steep gradient of polarity (from 18 to lOOo/o of wild-type activity) described by z mutations in the quarter of the gene closest to y reflects the distance of the non-

338 A. NEWTON

sense triplet from the more efhcient start signal at the beginning of the y message.

Left unexplained is the extreme polarity caused by nonsense mutations located in the beginning of z. Perhaps start signals are not present in this portion of z or, alter- natively, an ef6cient signal is required for continued transcription and translation after chain termination in the early part of the lac messenger.

The above interpretation supports the view that the primary defect in polar nonsense mutants is a translational one. Polarity would result from premature chain

termination at a nonsense triplet in a region of the messenger where efficient re-initia- tion of polypeptide synthesis is not possible. Two findings in the lac operon are consistent with this. First, addition of an operator-distal deletion which covers both NG125 and YA596 (Fig. 1) to U239 increases the transacetylase activity by over threefold (Newton, 1966). Second, the extreme polar mutation, 2, reverts to almost wild-type expression of galactosidase permease activity by the insertion of an extensive operator-distal deletion which does not delete the original mutation (Beckwith, 19643; Newton et al., 1965). As previously suggested, the deletions would suppress polarity by placing the site of chain termination closer to the efficient start sequence for the y messa#ge. How the interruption of translation leads to a reduction in the levels of messenger RNA in polar strains is not known (Contesse, Naono 8z Gros, 1966) ; models which involve messenger destruction (Beckwith, 1964a) and messenger synthesis (Stent, 1967) have been proposed.

Results reported by Michels & Zipser (1969) on double nonsense mutants of lac also indicate that x contains sites for re-initiation of polypeptide synthesis.

I am indebted to Mrs Willene Wright and Mrs Elaine Kates for assistance in construction of the double mutants used in this study.

This work was supported by U.S. Public Health Service Grant no. 14622 and Bio- chemical Sciences Support Grant no. FR-07057.

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