Mapping of polypeptide reinitiation sites within the β-galactosidase structural gene

J. Mol. Bid.

(1969) 41, 341-347

Mapping

of Polypeptide Reinitiation Sites within the fi-Galactosidase Structural Gene CORINNE A. MICHELS AND DAVID ZIPSER Department

of Biological

Sciences

Columbia University New York, N. Y. 10027, U.X.A. (Received 26;Augzcst 1968,and &‘-revised form 22 January

1969)

A method is described for mapping reinitiation sites within structural genes. The mapping techniques are used to demonstrate the presence of two such sites in the z gene of the luc operon. The results suggest that the reinitiation sequence is not AUG acting alone.

1. Introduction Polycistronic messenger RNA codes for the synthesis of several distinct structural proteins. This implies the existence of delineator nucleotide sequences that direct termination and reinitiation of polypeptide synthesis. The nature of these delineator sequences is a central problem in the complete elucidation of the genetic code. While logically it is not necessary to have a different nucleotide sequence for termination and reinitiation, experimentally it has been found that these two functions can be performed separately. The codons UGA, UAG and UAA lead to polypeptide chain termination (Sarabhai, Stretton, Brenner & Bolle, 1964; Stretton & Brenner, 1965; Brenner & Beckwith, 1965; Sambrook, Fan & Brenner, 1967; Zipser, 1967b). (In this paper we will use “terminator” when referring to these codons rather than “nonsense,” the historical term.) The codons AUG, GUG and perhaps GUA have been shown to initiate polypeptide synthesis, at least in in vitro systems when their position in the polyribonucleotide message is at or near the 5’-terminus. When AUG, GUG and GUA are in internal positions and preceded by an initiator, they code for methionine, valine and valine, respectively (Clark & Marcker, 1966; Webster, Engelhardt & Zinder, 1966; Adams & Capecchi, 1966; Ghosh, SolI& Khorana, 1967). Despite the fact that codons having delineator functions are known it has not yet been shown that any of these triplets is actually used alone to initiate or terminate any wild-type proteins in vivo. The study of the polypeptide terminator codons has been greatly facilitated by the fact that when such codons occur within a structural gene the protein fragments made are invariably lacking in activity and thus the terminator codon appears as a mutation. The situation with the polypeptide reinitiation sequence is quite different. Reinitiation sequences have been generated by mutation both in the gene specifying the rII protein of T4 (Sarabhai 8t Brenner, 1967) and the gene specifying the ,6galactosidase of Escherichia coli (Grodzicker & Zipser, 1968). In the case of ,$-galactosidase the reinitiation mutation by itself produces a protein indistinguishable from wild-type. Thus, reinitiation sequences occurring within a, structural gene need not, 341

342

C. A. MICHELS

AND

D. ZIPSER

in general, be mutants but can have a wild-type phenotype. Presumably it can also have a mutant phenotype. Since reinitiator mutations with wild-type phenotype have been found it is clear that reinitiator sequences may occur naturally within structural genes. In this paper we describe a technique which makes it possible to map the position of naturally occurring polypeptide reinitiation sequences within a structural gene. Henceforward we will refer to polypeptide reinitiation sites with the Greek letter rr. The procedure for mapping m is based on the observation of Yanofsky & Ito (1966) on the polarity of double terminator mutants. (For a discussion of the relationship of polarity to terminators see Newton, Beckwith, Zipser & Brenner, 1965; Newton & Zipser, 1967.) Yanofsky & Ito (1966) found that when two terminator mutants were put in the same cistron the polarity of the double mutant was the same as that of the first member of the pair (i.e. the most operator proximal). However, when the two terminator mutants were separated by a cistron boundary the pair was considerably more polar than either member. Some property of the cistron boundary allows the second terminator mutation to express its polarity. Clearly it is reasonable to assume that the polypeptide reinitiator sequence (v) within the cistron boundary gives rise to this effect. Thus, the degree of polarity of double polar mutants within a single structural gene serves as an operational test for the presence of r. Results of experiments with pairs of terminator mutants designed to locate polypeptide restart sequences in the lac operon are presented here. In addition to conflrming the result of Yanofsky & Ito (1966) concerning the effect of normal cistron boundaries on pairs of terminator mutants, we also demonstrate that there are two highly efficient reinitiation sequences (r(p) and T(W)) within the structural gene for /Lgalactosidase. The significance of this result is discussed with respect to the nature of the polypeptide restart sequence and the position of the ~(13) and n(w) sites in relation to complementation within the x gene.

2. Materials and Methods (a) Bacterial

strains

The mutants used were isolated by Zipser (19676). To obviate errors introduced by secondary mutations in the genetic background, each lac- mutation was transferred into the prototroph strain MO, an F- which is streptomycin resistant. The terminator codon designation of each strain is given in Table 1. To construct doubles between a pair of markers, F’Zac heterodiploids between the pair were constructed. If the pair complemented, the doubles were obtained from cured Zachomogenote segregants. If the pair did not complement, the doubles were selected from among the cured Zac+ reoombinants. This latter technique takes advantage of reciprocal recombination (Herman, 1965). In both cases the frequency of doubles was in the range of 10-l to 10-Z. (b) Determination To determine acetylase level Zipser, 1969).

of appropriate

wild-type

levels

accurately the degree of polarity of a given mutant was established by reversion techniques described

its potential previously

wild-type (Michels &

(c) Growth of cultzLres The cultures were grown in Casamino acids medium. The medium contains 1% Casamino and 5 x 10m4 &I-isopropyl-fi-D-galactioside in M9 (see Sarabhai & acids, 1 mM-MgSO,, Brenner, 1967). The cultures were inoculated from a fresh broth culture grown to saturation overnight without shaking at 37°C. The inoculum was diluted SO-fold into Casamino

POLYPEPTIDE

REINITIATION

SITES

TABLE

WITHIN

z

343

1

Terminator codon designation of terminator mutants

Strain

Terminator codon designation UAG UAG UAG UAG UAG UAG UGA UAG UAG UAA UGA UAG

NG545 NG624 NC422 NC608 NGlOOO NG1012 NG813 NG200 NG699 NG659 NG745 NG707

The terminator codon designation of each mutaut (except NGIOOO) was to its suppression pattern by methods described in Newton et al. (1965) Mutant NGlOOO required the isolation of a new suppressor strain which when & Brenner, 1967) with known T4 terminator mutants was showu to be an

acids medium and grown are dividing exponentially.

determined according and Zipser (1967a,b). spot tasted (Sarabhai amber suppressor.

with vigorous shaking for 4 hr at 37°C at which time the cultures The doubling time under these conditions is about 55 to 60 min. (d) Transacetylase

assay

The percentage transacetylase protein synthesized by each strain according to the procedure described in Michels & Zipser (1969). (e) Mapping

was

determined

of mutants

The order of the mutants in Figs 1 and 2 was determined by deletion mapping methods described previously (Newton et al., 1965). The mutants NG813 and NG1012 are UGA and UAG, respectively, and are in the same codon (Zipser, 1967a).

3. Results Strains containing two lac - terminator mutants were constructed and their rates of transacetylase synthesis determined. The results are shown in Table 2. The doubles fall into two classes. In class I, the introduction of the second, more operator distal, terminator mutant had no marked effect on the polarity of the strain. These results are comparable to those obtained by Yanofsky & Ito (1966) for double mutant strains in which both terminator mutants were in the same cistron of the tryptophan operon. In class II, the double mutant strain was more polar than either mutant alone. There are two kinds of class II strains : the z--z and x-y pairs. The increase in polarity of all x-y pairs is to be expected since these mutants are in different cistrons. The increased polarity in certain pairs of z--z doubles indicates the presence of hitherto undetected polypeptide reinitiation sequences. A comparison of the z-z doubles which fall into class I to those which are in class II clearly shows that the increase in polarity is not directly related to the specific terminator mutants used in the doubles but requires that specific sites in the z gene map between the two mutants of the double.

C. A.

344

MICHELS

AND TABLE

Transacetylase

ZIPSER

2

of double terminator

mutants

Transacetylase protein (percentage of wild-type) First Second Double mutant mutant

Strain

Class I: z-z double NC545 -NC*624 NG422 -NC608 NC422 -NC*1000 NC1012 -NG200 NG200 -NC699 NC200 -NG659

activity

D.

(2)* (1) (2) (2) (1) (3)

3.7 4.4 4.4 7.1 16.8 16.8

4.7 7.1 12.9 16.8 51-o 86.5

3.3 2.9 3.0 6.7 20.8 20.1

Class II: z-y doubles NG624 -NG707 (1) NC200 -NG707 (3) NG813 -NG745 (1)

4.7 16.8 6.3

23.7 23.7 4.3

1.7 11.0 0.5

Class II: NG624 NG624 NG624 NG422 NC608 NO608 NGIOOO NC1000

4.7 4.7 4.7 4.4 7.7 7.7 12.9 12.9

7.7 7.1 16.8 7.1 7.1 16.8 7.1 6.3

1.4 0.8 1.2 1.0 2.3 2.7 3.2 3.0

z--z doubles -NC*608 (1) -NC*1012 (2) -NG200 (4) -NG1012 (3) -NG1012 (2) -NC*200 (1) -NG1012 (3) -NC813 (2)

The level of thiogalaotoside transacetylase protein synthesized by the double terminator strains is compared to the levels synthesized by the single terminator strains. First mutant indicates the more operator proximal mutant of the double. Second mutant indicates the more operator distal mutant of the double. *Indicates the number of separate isolates of the same double mutant used to determine the level of transacetylase protein.

The data is consistent in the sense that an unambiguous map of the position of the m sites can be constructed. Such a map is presented in Figure 1. The entire region between NG545 and NG659 has been surveyed for reinitiation sequences. This region represents all of the x structural gene with the exception of short segments at either end. Only two reinitiaton sequences have been found. One maps between NG624 and NG422, and the second maps between NGlOOO and NG1012 (NG813). For reasons which will become clear below, we shall use the notation ~$3) and r(w), respectively, in referring to these two sites.

4. Discussion We have found two polypeptide reinitiation sequences, 7r(/3) and n(w) within the z structural gene. The identification and mapping of these sites was carried out using double polar mutants. Following the work of Yanofsky & Ito (1966), we concluded that an initiator for polypeptide synthesis was located between two polar mutants if the double mutant was much more polar than either of the single mutants alone. This criterion led us to an unambiguous map of the z gene showing the positions of two polypeptide initiation sites, r(p) and T(W) (Kg. 1). The data indicate that these reinitiation sequences are located in specific small genetic regions and are not spread

POLYPEPTIDE

REINITIATION

SITES

‘WITHIN

345

z

out smoothly over the map. This suggests that reinitiation is occurring at a limited number of sites (probably two) and is not due to a large number of sites. We, of course, have not ruled out the existence of many other very weak reinitiation sites. ~$3) and Z(W) cannot be transcription initiators as this would lead to constitutive synthesis of the distal enzyme which is not observed. z

Y

a ---

HG545 I

NGb24 I

i

1

0

“bJ)

ff(p) NG422 Nt608 I I

NG813 lNG745. NGIOOONt1012NG200NC699NG659 NG707) ! I I 1 I / I

---

I

‘I

cm-----, &----------+ + -----

I

----

k---4

Class I

---,

+------+

ClQSS n (Z-Z) &----, )----------+ L----I

FIG. 1. Mapping of terminator mutants in the Zac operon and a diagramatic representation of the double mutants. The map is diagramatio and does not indicate actual genetic distance. The figures above the map are: o, operator; z, the structural gene for @-galaotosidase; y, the structural gene for fl-galactoside permease; a, the structural gene for thiogalactoside transacetylase; Z-(B) and m(w), the naturally occurring sites for the reinitiation of protein synthesis described in the text. The lines below the map indicate the region of the map which lies between each double. The solid lines indicate those doubles which fall into class I (see Table 2) i.e. a rr site does not lie between the mutants of the double. The broken lines indicate those doubles which fall into class II (see Table 2) i.e. a rr site does lie between the mutants of the double.

Two, and possibly three, codons have been shown to code for chain initiation with N-formylmethionine in vitro. One of these codons, AUG, is the only known internal codon for methionine. The structural gene for ,L?-galactosidase codes for 24 methionines (Craven, Steers & Anflnsen, 1965). If AUG alone were sufficient to code for efficient restarting one would expect to find an absolute minimum of 24 polypeptide restarts within the structural gene for ,%galactosidase. This does not include GUG and out-ofphase initiators. In the experiments described here only .two restarts were found. We feel this is a very significant discrepancy and propose the following set of explanations : (1) All the methionines in z are grouped at the ends and in the two restart regions. (2) There is an additional, as yet unknown, internal codon for methionine. (3) It is not the reinitiation sequence but some other feature of cistron boundaries that gives rise to the Yanofsky-Ito effect. (4) AUG alone is not sufficient to act as a reinitiator in viva. We favor number 4 as a working hypothesis for the following reasons. Studies of the cyanogenbromide peptides of purified /Lgalactosidase (CNBr breaks polypeptide chains at methionine residues) have shown that 20 to 25 peptides of varying molecular weight are produced (Craven et al., 1965). There is no indication in these results that the methionine residues are concentrated in small regions of the protein. Another internal codon for methionine would conflict with existing in vitro and in vivo evidence on the code (Tsugita & Fraenkel-Conrat, 1962; Funatsu & Fraenkel-Conrat, 1964;

346

C. A.

MICHELS

AND

D.

ZIPSER

Weigert & Garen, 1965; Yanofsky, 1965; Yanofsky, Ito & Horn, 1966; Wittmann & Wittmann-Liebold, 1966). While the structure of the cistron boundary is completely unknown, making it impossible to rule out number 3 as an explanation of our results, we feel that number 4 is a less complex working hypothesis. If AUG alone is not sufficient for initiation then: (1) a specific base or sequence of bases adjacent to the N-formylmethionine codon or; (2) a specific secondary structure at the site of the initiation codon may be required. The gradient of polarity as described by Newton et al. (1965) in the z gene of the lac operon and by Yanofsky & Ito (1966) in the tryptophan operon demonstrates that the degree of polarity of a terminator mutant is dependent upon its location in the cistron: the greater the map distance between the terminator mutant and the next cistron boundary, the more polar the terminator. In addition, it appears that a new gradient of polarity starts at each cistron boundary of the operon. The present site of the theories of polarity propose that it is the distance from the reinitiation cistron boundary which is the significant factor in determining the polarity of a terminator mutant (Newton & Zipser, 1967; Sarabhai & Brenner, 1967). If so, why has only a single gradient been demonstrated in the z gene and not the three which would be expected based on the efficient reinitiation of polypeptide synthesis at V(P), n(w) and the z-y cistron boundary? In fact, there is some hint in the data given here of a discontinuity in the gradient near NGlOOO. NGlOOO is significantly less polar than the mutant which maps closest to it on the operator distal side. A consideration of the map position of NGlOOO in relation to CT(W)suggests the existence of a discontinuity in the gradient in this region of the map similar to the one found at the distal end of z at the z-y cistron boundary. The gradient of polarity must be clarified before an absolutely unambiguous interpretation can be given to ~(,8) and n(w). Work is now in progress in our laboratory to reinvestigate the fine structure of the gradient of polarity in the z gene of the lac operon. The preliminary results of this work have shown that there are in fact three gradients of polarity in the z gene as expected if z-(p) and r(w) are polypeptide reinitiators. When this work is completed, it should give some independent evidence of the nature of n(p) and V(W). The work The question arises as to how efficient n(P) and T(W) are as reinitiators. described here gives no good way to determine this. However, since the polarity effect of ~(/3) and n(w) is about the same as that of z-y boundary, it seems reasonable to assume that the internal reinitiators are at least as efficient as the normal initiator for permease. In extracts from z deletion strains, two peptides (called a and w) which complement with extracts of x point mutations have been isolated (Ullmann, Perrin, Jacob &: Monod, 1965; Ullmann, Jacob $ Monod, 1967). Figure 2 presents a map of the z gene and indicates the portions of the map specifying the a and the w peptides as defined by Ullmann et al. (1965,1967). In addition Figure 2 shows the location of r(p) and n(w) on the complementation map. n(p) is located to the right of the boundary between the a-specific segment of the z gene and the remainder of the map. Because of the extreme polarity of terminator mutants in the a-specific segment, it is not possible by the techniques described here to determine if a third r site is located at the a complementation boundary. It is clear, though, that the T(P) site defined here and the a-complementation boundary defined by Ullmann et al. (1967) do not coincide.

POLYPEPTIDE

REINITIATION

SITES

z

de ---

%4

NC624 I

“131 l:rNGs4s !/

I

III

NC,422 I I

I I

I

NGl2S I

.z

I

“W 0

WITHIN

X62 YAS36 Nt606 I

YASS9 WI000 I

347

Y

=

1

NGlOl2 NGBI3

I 1 NG200

III

Nt699 I

I’

”

NG 659 I

I

---

I

I OJ /3 Fra. 2. A comparison of ?r(/3), r(w) and the complementstion boundaries in the z gene. As in Fig. 1, the above diagram indicates the order or the z- mutants. In addition to the mutants used in this study, those used by Ullmann et al. (1965,1967) in characterizing the a and w peptides of /?-galaotosidase are shown. The positions of T@) and v(w) are indicated by the solid vertical lines. The broken vertical lines define the portions of the z gene specifying the x) and o peptides. j3 indicates the remainder of the map specifying other, as yet unoharacterized, peptide( 05

1

n(p) does not map at the beginning of the region specifying strong w complementation but seems to be slightly more operator proximal in the region of weak w complementation. More information concerning the mechanism of complementation in ,B-galactosidase and a more detailed analysis of initiation at ~(/3) and n(w) are necessary before the relationships, if any, between the complementation boundaries and the CT(~) and V(W) sites can be understood. This work has been supported by the National Science Foundation (Grants GB-7127 and GB-3231) and the United States Public Health Service (Grant 5 ROl-GM-14676-01). One of us (C. A. M.) is supported by a National Science Foundation Predoctoral Fellowship. We would like to thank Mrs Hallie Phillips and Mrs Josephine Collins for their technical assistance and Mr Sandy Zabel for mapping the z- mutants. REFERENCES Adams, J. M. & Capecchi, M. R. (1966). Proe. Nat. Acad. Sci., Wash. 55, 147. Brenner, S. & Beckwith, J. (1965). J. Mol. Biol. 13, 629. Clark, B. F. C. & Marcker, K. A. (1966). J. Mol. Biol. 17, 394. Craven, G. R., Steers, E. & Anfinsen, C. B. (1965). J. Biol. Chem. 240, 2468. Funatsu, G. & Fraenkel-Conrat, H. (1964). Biochemistry, 3, 1356. Ghosh, H. P., S611, D. & Khorana, H. G. (1967). J. Mol. BioZ. 25, 275. Grodzioker, T. & Zipser, D. (1968). J. Mol. BioZ. 38, 305. Herman, R. K. (1965). J. Bad. 90, 1664. Michels, C. A. & Zipser, D. (1969). Biochem. Biophys. Res. Comm. 34, 522. Newton, A., Beckwith, J., Zipser, D. & Brenner, S. (1965). J. Mol. BioZ. 14, 290. Newton, A. & Zipser, D. (1967). J. Mol. BioZ. 25, 567. Sambrook, J. F., Fan, D. P. & Brenner, S. (1967). Nature, 214, 452. Sarabhai, A. S. & Brenner, S. (1967). J. Mol. BioZ. 27, 145. Sarabhai, A. S., Stretton, A. 0. W., Brenner, S. & Bolle, A. (1964). Nature, 201, 13. Stretton, A. 0. W. & Brenner, S. (1965). J. Mol. BioZ. 12, 456. Tsugita, A. & Fraenkel-Conrat, H. (1962). J. Mol. BioZ. 4, 73. Ullmann, A., Jacob, F. & Monod, J. (1967). J. Mol. BioZ. 24, 339. Ullmann, A., Perrin, D., Jacob, F. & Monod, J. (1965). J. Mol. BioZ. 12, 918. Webster, R. E., Engelhardt, D. L. & Zinder, N. D. (1966). Proc. Nut. Acad. Sci., Wash. 55, 155. Weigert, M. G. & Garen, A. (1965). Nature, 206, 992. Wittmann, H. G. & Wittmann-Liebold, B. (1966). Cold Spr. Harb. Symp. Quant. BioZ. 31, 163. Yanofsky, C. (1965). Biochem. Biophys. Res. Comm. 18, 898. Yanofsky, 6. & Ito, J. (1966). J. Mol. BioZ. 21, 313. Yanofsky, C., Ito, J. & Horn, V. (1966). CoZd Spr. Harb. Symp. Quunt. BioZ. 31, 151. Zipser, D. (1967a). Science, 157, 1176. Zipser, D. (1967b). J. Mol. BioZ. 29, 441. 24