J. Mol. Bid.
(1978) 125, 407-G%
Synthesis and Degradation of Termination and Premature-termination Fragments of /?-Galactosidase in Vitro and in Vivo
(Received 13 September 1977, a,td in revid
,fornt
4 July
1978)
A rrumbcr
of abnormal polypept,ides whicl~ HI‘ a11 t,arly amber mutation in the 2 gene, is degraded iI1 ttle in vitro system. The rnwhanism of degradation appears t,o be specific for small abnormal polypeptides. 1nttwral wmitiation polyprptides generated by nonsense mut,ations, which have hwrl found in viva. are not detrct,ed irl thrl i?, vitro protein synthesis system urrdrr t,lrv conditions 71~4 IIerc. /Cscherich~ia
1. Introduction Mher mutzitions or mistakes which occur during transcription or translation oan lead to the synthesis of abnormal polypeptides. Polypeptides resulting from mutations can be of several types. Nonsense mutations produce protein fragments terminating at bhe altered codon. In addition. certain nonsense mutations result in the synthesis of int’ernal reinitiation polypeptides. due to initiation at a site downstream from the nonsense mut’ations and termination at the normal t)ermination site. DeleCon mutants can give rise to a number of types of fragment. Internal deletions of some multiple of t’hrre nucleoCdes (in-phase deletions) produce fragments with normal ends, but with amitm acids deleted internally. Internal deletions of a non-triplet number of nucleoticks result in bermination at an out,-of-phase triplet, and in some cases also generate
;Q 1978 Academic
Press Inc. (London)
Ltd.
408
.I. 1.. \l=\SLKY
internal reinitiation fragments. Prematurr-trrmillirtion fragments aIso t)r earlv quit,ters art’ readily seen in the products of protein svnthesix in vitro (Atkins &, (A~steland. 197.5: Chambers & Manley, 1973: Dottin rt al., 1975: Kerr et al.. 1972: At’kins et al.. 197.5). The view has been that this is an arbifa,ct of the in Gtro systems. However, if premature termination does occur frequently in viva. it might not IW apparent for at least two reasons. First, abnormal polypeptides. including prematurcbt,ermination fragments which result from nonsense mutations, are trrquently rapidly and specifically degraded in viwo (for review . see Goldberg & Dicta. 1974). Nothing is known about the mechanism(s) by which these polypeptides are recognized and then degraded, but several mutants deficient in degra,dation in ciao have been isolat,ed (Bukhari & Zipser, 1973 ; Apte et ul.. 197.5). This t’ypc of specific degradation has not lxw~ demonstrated ire Second, such premature-termination fragments would be likely to be difficult to det,ect hy standard biochemical t,cxchniqut:s. It is likely that these molecules would he lost, by routine purification schemes. However, w&in techniques might be expected to uncover such polypeptidrs, if they exist. ITor example. a,ntibody precipitation followed by sodium dodecyl s~~lfatc?/pot~~~~cr~lamide gel elect’rophoresis of the precipitated proteins should revt~al any ~~~~c~~i~~t~n~~f~-tarrnillntion fragments which contain exposed ant,igenic drt,erminant,s. in this paper 1 report the charactrrizatiori of sc:vt>ral types of abnorma t polypeptides synthesized CAL and in ‘ciao. The potypeptides synthesized from the % gene of the lac operon of Escherichia coli. which encodes ,B-galactosidase. have been purified by antibody precipitation and analgzed on SDS~/polyacrplamide gels. 1 describe a set of specific protein fragments. and provide evidence that they are dutt to premature t,ermination of protein synthesis within thtt Z gene. These fragment,s art’ vitro. The total amount of these fragments is estimated to made in. viva as well as hr at least 23O!;, of the t,otal B gent’ protein. These results show that, premat,uretermination, at least, in the case of /%galactosidasc, is not an artifact of protein synthesis i?z vitro, and suggest that synthesis of aberrant polypeptidrs may he much more common than previously thought. Fragments synthesized vitro from DNAs containing amber mutations have also been examined. They are identica,l in size to the corresponding fragments made in l!ivo. However, unlike many nonsense fragments synthesized in 1~it10~ which are rapidly degraded, the fragments synthesized in vitro. with one exception. arc very Aable. The exception, a fragment produced by an early amber mutation, cannot be detected k)y SDS-containing gel electrophoresis, and evidence is presented that, the IMP TLOUO synthesized mutant polypeptide is specifically degraded itb vitro. (‘il,ll
vitro.
vitro
it,
it1
2. Materials and Methods (a) Materials Rabbit arlt~i-8-galactosidase autisenlm UXR a gift from 31. Zipser. It, was pro-adsorbed \yith an equal voh~rnc of a cortcentratcti ext-ract made frown a lac deletio!l st.rain (strain 1 in ‘Fable 1) by incubation in the cold for 3 to 4 h. In one case t,he i~rl~nllno~lobulin fraction was purified as described by Atkins & Gest,elanp (1975). [35S]methionme (200 to 400 Ci/mmol) was either from New England Nuclear. Bost,on, Maas. or made as drscribed by t Abbreviations used: SDS, sodium dodecyl sulfate; CAY, cat,abolite act,ivator protein
TII-Tris (pH 7,J-). 200 IIIRI1 RI-urea, and 1 l/o Tritorl X100). or, in some earlier c~xperirnor~ts (tlrc KCl, 1 mrvr-MgCl,, results of one of these are sl1own ill Pig. 5), with 1 ml of buffer 13, wliiell is k)llffiar CTxvit~llolct. tho urea and Triton X100. The addition of I >I-urea plus 1 “() ‘I’ritoti S IO0 to t I)(’ ar~t~rl)oti~~ precipitation buffer, a modification of a tt~chniquc~ originally drscribrd t,y Oberg et a/. (1975), was extremely effective in reducitlg background noll-specific l”.f.‘,il.)itatiotl. I IIsoluble material was removed by centrif1~gat,ion at 30,000 g fi~r 15 IIlirl. (f) Protein
synthesis
in vi\-0
Cells were grown in 10 ml MQ medium (Miller, 1972), wit11 glycerol as tile carbon source. at 34°C. Any required ammo acids (see Table 1) were u,ddcd at, a concn of 50 &ml. ‘lYLc cells were grown until they reached a Klett reading of -85. at which t,imc 5 x 10F4 &Iisopropyl-thio-/3-D-galactoside \\-a~ added t,o arly cultures tlot const itutivr for tllrx (1xpression of the Zac operon. One minute later. 100 &i of 135S]Inet’lliorline w(‘re added and the cells were allowed to grow for 3 more min, except where noted. They were then harvested on ice, washed with 2 ml buffer H, and rcslispended in I ml huffkr C. The cells were lysed by sonication in an ice-bath for 30 s wit,h a Hiosonic IV sonicator. Debris was removed i,v centrjfugation at 30,000 g for 15 min. Roughly BOY/, of thra added radioactivit~y was recovered as trichloroacetic acid-insoluble polypeptides. (g) Analysis
of radioactive
1acZ gene-specijicd
polypeptides
Samples were routinely analyzed by precipit,ation with anti-p-galactosidaso antiserum, followed by SDS/polyacrylamide gel electrophoresis. This was done in or~e of two ways. For all the in vitro samples, and those in viva samples not, constitutiva for lac operon expression, approx. 1.5 pg of unlabeled fi-galactosidase was added to the 30,000 g supernatant fraction, to serve as carrier for the antibody precipitation. (TIE carrier was added as a crude extract, prepared from strain 3 in Table I, essentially as described in the preceding section except that no label was added.) A sufficient amount of antiserum was added to precipitate all the carrier, and reaction mixtures were incubated at 0°C for a minimum of 3 h, but usually overnight. The amourlt of /3-galactosidase-related polypeptides in the labeled extracts (in pg), is so small that, it does not affect the antibody titratioll. No differences were ever observed when precipitation was performed for different, lengths of time within this range. Three samples were prepared from strains constitutivr for expression of the lac operon (strains 12 to 14 it1 Table I ). In tile in viz10 samples prepared from these strains, the amount of /3-galactosidase-related polypeptides may not, be insamples. significant compared to tile 1.5 pg of carrier, a.s was the case with t,he prcviolls In the case of strain 12, which is wild-type, the concentration of ,6-galactosidasc can be determined by enzyme assay. A volume of sample which corresponds to 1.5 fig of 8. galactosidase was removed from bhe 1 ml of supernat,ant described in the preceding section (this is roughly 50 pl), diluted to I ml with buffer C, and precipitated directly
TERMINATION
FRAGMEKTS
OF p-GALACTOSIDBSE
411
with antiserum. For strains 13 and 14, I determined in preliminary experiments how much antiserum was necessary to precipitate l-5 rg of carrier in the presence of an arbitrary amount (100 ~1) of each of the 2 mutant extracts. This amount of antiserum was then used to precipitate 1.5 rg of carrier plus 100 ~1 of each labeled extract, in 1 ml of buffer C. In all the samples, precipitates were collected by centrifugation at 20,000 g for 10 min, washed 2 x with buffer C, and dissolved in 50 ~1 of sample buffer described by Anderson et al. (1973) (0.08 M-Tris (pH o-8), 0.1 M-dithiothreitol, 2% SDS, 10% glycerol, 0.004”6 bromophenol blue). The samples were boiled for 2 to 5 mm, and a sample (5 to 25 ~1, depending on the sample and the experiment) was loaded on a loo/ SDS/polyacrylamide gel, which was made, and run, stained and destained according t,o Anderson et al. (1973). At this point the samples could be compared to determine if the efficiency of antibody precipitation was the same in all cases, by comparing the intensity of t,he stained flgalactosidase carrier bands. Part of a typical stained gel is shown in Fig. 2(b). Gels were then dried under vacuum and exposed to Kodak X-ray film SB-54, usually for about 1 to 3 days. When quantitation of band intensity was required, autoradiograms were scanned with a Joyce-Loebl Chromoscan. and the peaks were integrated with the aid of a Numonics planimeter. (11) Auto- a complemenlation
assay
Auto-cc assays were performed as described by Morrison & Zipser (1970), except that in all cases the auto-a donor was a loo-p1 protein synthesis reaction mixture diluted to 200 ~1 with buffer D (0.02 M-Tris (pH 7.2), 0.2 M-p-mercaptoethanol, 0.04 M-EDTA, 0.01 XNaCl).
3. Results (a) Comparison
of in vivo and in vitro synthesized polypeptides
of several mutations which result in the production of polypeptide fragments in vivo are shown. These fragments lack enzymatic activity, are of lower molecular weight than the wild-type protein, and are degraded in vivo with half-lives as short as five minutes (Goldschmidt, 1970a; Bukhari. 1975; Manley, unpublished results). To study the synthesis of the fragments in vitro, these various mutations were transferred to Xpluc5 transducing phage by B. Apte & A. I. Bukhari, who generously provided them for these experiments. The phage DNA was used to direct the synthesis of proteins in vitro, as described in Materials and Methods. Z gene-related polypeptides were purified by antibody precipitation and analyzed by electrophoresis in SDS/polpacrylamide gels (see Materials and Methods). Figure 2 compares in vitro synthesized polypeptides and the corresponding in viuo Figure
1 is a map of the Z gene of the lac operon of E. coli. The positions
Fro. 1. Genetic map of the ZacZ gene. The numbers above the line represent amber mutants. x( 1) is a mutation which results in the synthesis of an internal reinitiation polypeptide in the double mutant 646rr( 1). The broken lines define the 3 complementation regions of the gene. The 2 enclosed areas underneath the line indicate deletions.
41”
.I . 1,. JI .\ s Lb: \
products. The mutant fragments synthesized ir/ vilro and it1 viuo wmigrate, providing strong evidence that the mut,ant pol,ypept,idcs produced it/ vitro aw t hc si~~~l(~;IH t host. synthesized in vivo. As equal amounts of each of the itl ~ifro samples UWY: loaded onto t,tic gel, the nnAar yield of the various % gene-derived polypeptides can be estimated. 1Cor cxamplr; the introduction of IO’-5. a mutation which eliminat,es the requirements for the catabolic~ activator prot,ein and cAMP for lac optwn transcript,ion (Silverst~onc it ccl.. 1970). into ApZac5 resuhs in a great increase in t,hc amount of /3-galactosidase synt,hesized it/ r!itrct (Big. 2 slots d and e). (The lvsogen from which this DXd \vas prepared \\w kindI> provided by P. Bazzicalupo.) Also Xplr~6 IT\:-5 DNA tlireot~s t,he synt,hesis of approximately ten times as much enzyme as does hpZnc.5 DXd (Eron & Block: 1971 ; set: also Materials and Methods). Thus, even though the S-30 extract used in t,hese experiments is CAP+, it, seems that activcb CAP protein is a limiting factor under t~hew conditions, which are optimal for a high yield of P-gaI~bct,osid~~s~. A comparison of ttw intensity of tha wild-type potypeptidc hand lwoduwtl t)y hpZac.5 DE\;;{ wit,h ttlc int)txnsities of the bands produced in vitro t)y tllcl various mutant, Dl\‘As sl~ows tllat’ iill tIlta as \\,($I1 i18 tmllc liolypel~tidc~ s,vnthesiz,cd nonsensf: mutant termination polypeptitlcs. from DNA containing the small in-phase dt~letion. A21 _ art present in at lcast 75’1, ttic protein yield. assuming an equal distribution of’ mcttrionim~ wsiduw throughout (Fowler $ &bin, 1977). This is in strwli cwnt,rast, t,o ttle yield 0litaincYl with tllc samf fragments synthesized it/ tlivrt. The nonsw~s~~ tc~rmination tiagmc~nts a~‘(’ recovc~retl in yields of at best, .W’;, of tlrc wild-t,ype lwc*l. we11 aftw ii pulse label. Other mutant, polypeptides that, one might, expect, to tint1 synthesized it/ citro are r~:c:ovwcd in veq low yields, if at all. The d&l fragment. \\ hich most prohalily results from an inphaw internal deletion, is recovered in very poor aii(l variable yield. TWO internal reinitiation polypeptides which can he detected it/ ri?vo. thaw from the mutat1t.s 2 t 77 (RS in Pig. 2) a,nd 54$7(l) (Zipwr ?‘I al.. IWO: Xpttb ct ~1.. 1975) (data for .‘,-l:?n( 1) not sho\vn), cannot, be (let&cd at all iI/ vitru under t,hcs wnditions usetl Ilcre. In addition, thtb 5457r( I ) nonsense termination fragment’s from t\\-0 carty % gvnc~ amtw mutations, (see Fig. 1) and @24(%v\-hichmaps M\vet~tl 54hr( I) and 2177). c:annot lie tletectt~d. This is true not only for the iti vitro products Ijut alsc~. ;\t~ lc,ast in that c*aso of’ 5457~(1). for the in vivo polypeptide (dat,a not sho~+n). l?inally, in addit,ion to the major component in c~ch sample. a number of ot’hrr usually bvith greater c,lcct~ophorc:tic mohilities. can tw tlet~ected in most polypeptides. of t,he samples.
(b) Lower wtolecdw
weight frqpetttn
What is the origin of these polypeptides. \\-ith apparent molecular weights less than unrelated to full-sized /3-galactosidase protomer? ‘I’ht?y could be, for example. /3-galactosidase and t,hus artifact,s of the antibody precipitation. The follo\ving rxperimerits show that at least nine are specific products of t,ht Z gene (indicated by solid lines in Pig. 2). slots a and 1) of Figure 2(a) are antibody precipitates of 135QImethiotiirie-labr.1~~1 (sxtracts. from both ill vitro and it), vivo. in uhich no Znc DNA sequences were present. None of the polypeptides present in t,hc sampk. w thatf do contain lac DlVA can be detected. (It should be noted that the amount of physical material precipitated is tht same in all cases, owing to the addition of a large molar excess of unlabeled wild-type b-galactosidase; see Materials and Methods.) But perhaps aggregation which results
a
bc
defah
i
ik
Imn
BG
7 0 9
-H
(a)
414
J. L.
MANLEY
in non-specific precipitation occurs before the addition of the unlabeled wild-type was checked in two ways. The carrier. Thus the specificity of antibody precipitation first shows that non-specific aggregation during preparation of the crude extracts (e.g. during sonication) does not occur at a level detectable by the subsequent’ antibody precipitation. Strains 11 and 12, which are isogenic lac deletion strains except that one contains a X prophage and the other a XpEac5 prophage. were grown as described in Materials and Methods. The cultures were divided in two, and half of each wa,s labeled with [35S]methionine for three minutes. Then a large excess of non-radioactive methionine was added to stop incorporation of label. Each labeled culture was then mixed with the unlabeled reciprocal culture, chilled on ice and processed as described in Materials and Methods. The results of the antibody precipitation are shown in Figure 3 (slots a and b). There are a number of specific polypeptides of lower molecular weight tha,n the full-size (135,000 M,) polypeptide in the sample prepared from the cells lysogenized with hpZuc5 (numbered 1 to 9), and these are absent in the sample from the cells containing the h phage. This argues that these polppept’ides do not result from artifactual aggregation during sample preparation. I next wished to determine whether synthesis of these fragments is regulated by the lac controlling elements, as would be expected if they are actually products of the Z gene. Strain no. 15, which contains a wild-type copy of the entire lac operon, and is therefore inducible for synthesis of the lac enzymes (Aplac5 lysogens do not contain a functional I gene and are therefore constitutive), was grown up in duplicate cultures. To one the gratuitous inducer of the lac operon. isopropyl-thio-/l-n-galactoside, was added when the cells reached a concentration of 5 x lOa/ml. Two minutes later, [s5S]methionine was added, followed five minutes la’ter with a large excess of unlabeled methionine. The cells were harvested after 30 minutes of additional growth. To the other cultures [YS]methionine was added first, followed five minutes later by the chase. One minute after this, isopropyl-thio$-n-galactoside was added. A sample was harvested a,fter two minutes, and the remainder after 35 minutes of additional growth. The samples were processed as described in Materials and Methods (carrier p-galactosidase was added to the sa,mple induced for only two minutes to equalize the total amount of enzyme in each of the three samples). The results are shown in Figure 3 (slots c. d and e). Some polypeptides are present in all three samples. However, a number of polypeptides are present only in the sample induced before addition of the label, and these therefore must’ be specific products of the 2 gene. A close comparison shows that the mobilities of t.hese polypeptides are the same as those of the proteins indicated by lines in Figure 3 (slot a). On the basis of these results, and other experiments discussed below, I conclude that the polypeptidex. numbered 1 to 9, are products of the 1acZ gene. The fragment indicated hp the broken line will be discussed below. The polypeptides numbered 4 and 5 are only occasionally resolved. For example in Figure 3 (slots a and e), the autoradiographic bands are not well resolved. whereas in Figure 2 they are. Note that slot e (Fig. 3) represents a 30-minute chase. I have consistently observed that there is no change in the intensity of any of the &c-specific bands during the course of such a chase. This result. which is consistent with in vitro results presented below, argues that the fragments do not arise by proteolytic cleavage during growth. Also, the fact that the fragments are produced in st,rain 15 shows that they are not a result, of t,he presence of t,hc h prophagc, which is present in the other ijr vi~io samples. The nine polypeptides indicated in Figurc 2 have the same mobilities as those in
7a-9-
FIG. 3. Autoradiograms of SDS/polyacryIamide gels which shoxv that p-galactosidase-derived fragments are not artifacts arising from the antibody precipitation reaction. Slots a to e, controls for specificity of antibody precipitation (see text for details). a, [35S]methionine-labeled polypep. tides precipitated with anti-/l-galactosidase serum from strain 12 (Inc+ ). b, [35S]methionine-labeled polypeptides precipitated from strain 11 (&UC). c, polypept,ides precipitated from strain 16, which had been labeled, chased, and induced for 30 min. d, same as c, except, that cells were induced for only 2 min. e, same as c, except that cells were induced before addit)ion of label and chase. The solid lines indicate &galactosidase-specific fragments. The broken line indicates a fragment,, also apparently /?-galactosldase-specific, which appears to be missing amino acids from the ammoterminal end rather than from the carboxy-terminal end like thr other fragments (see text,). f to g, [35SJmethionine-labeled polypeptides synthesized in vitro and analyzed without antiserum purifioation. Immediately after the DNase step (see Materials and Methods), 1 ml of ice-cold 80% acetone was added to each sample. The precipitated protein was collected 10 min later by centrifugation, dissolved in 100 ~1 of gel sample buffer, and heated t,o 100°C for 2 to 5 min. A tot,al of 5 ~1 of each sample was then loaded on an SDS-containing gel. The templates were: f, X DN.4; g, hpZac6 UV-5 DNA. Lines indicate detectable fi-galactosidase fragments.
Il(i
.I. I,. 11:\ s 1,13Y
Pigurc: 3 and are present in both the i//, V;WJ and j/l ~?ifr~ sillrlplt’s. Ho\~c~vt~~~.i\ ~l~ltr~l)t~r of ~loll-spe~iti~ polypeptidrs t,hat, vary in I)otlr amount iit>(l tlurrll)c~r iI1 t hc* (lifY(*r(tnt samples il1x-L also apparent. lqor ~~xampk~. ill I~iyurc~ ;I (slot a) tlrcw iiu~ virtikally 110 non-specific polypeptides. but in t,lrcssamples in l~‘igurc~ 2 tllt’l’(’ ill’ the following results. The kinetics with \vhich th(a fragments appear. as assayed on SDS-containing gels, can provide some information as t)o how they arc fortncd. For example, in the case of wild-type /3-galactosidase, if the final step in the formation of the fragments is the same as it is for t,hc full-size prot,omer. i.c. tc~rrnination of th(b polypeptide chain, then one would expect to SW t,hcs c*oncc~ntration of t hc fragments increasing at the same rate as the concentration of the 135.000 LIJ~protomor. Howcvrr. if Ohere is an additional cleavage step in thr formation of t,htl fragmtbnts. t)hcn out might expect the concentration of bhe fragment’s to incrcbase only after a lag. when cornpared to the complete polypeptide. Figurt, 1 sholvs the products of protein synthesis in vitro directed to hpZac5 1’\‘-.5 DNA. for various timrs of synthesis ranging from five to 50 minutes. The P-galacl-osidasc~-~~l~lt(~(~ fmgrncnts (solid lines) do not sho\v a lag in appearance compared to \vild-type /?-ga,lactosidase. I II fact. itt bll(* (xarlip::t t’ime point at which t,he 135.000 ;I/,. pol?peptidc (*a111~ dt%rct,tbd (7.5 tnin) many of the smaller polypeptides cau also I)t: detc~ct~ed. at roughly t,hta sam(’ intensity ah thcb wild-type. However, at the conclusion of the reaction t,he intensity of the band produced by the full-size polypeptide is of the order of tcu times greatjcr than that of any of the fragment bands (see also Table Z), so it appears that the fragmc>nts are synthesized at! a greater rate. compared to t hc full-size protein. early in t’hcl reaction. \;c’h~ this is so is not, clear, but these kinetics of appearance provide strong c~vidcncc~that the fragments do riot arise by proteolytic clraragc of filll-sized ,Qalac*tosidast~. Thrrt: is t)j, one fragment, however. that’ appears with kinetic s consistent with it. originating degradation of a larger polypeptide. This fragment is shown hy a Ijrok(bn lint, in Bigurt~ 4, and is not, one of the polypeptides indicated it1 Figures 2 and 3. Another way to ask whet’hcr or not. th(b fragments ar(~ genrrattad I)y post-translational cleavage is to see if fragments continutb to hc generated afttxr protciu synthesis is halted. In the experiment sho\z,tr in Figure 5. various Xplac5 DNA wart’ used to direct protein synthesis in the in vitro sy&:m for 20 minutes. Than ~~llorwlnpht,l1icol
ii
(69)
R!) (30)
70 (61)
15 (36)
1.5
I .!I
3.1,
i.1,
I.4
I.li
I.0
4.0
s.9
6.6
13 16
1 .!I
4.7
x.2
ti.ti
2.4
I .:
. (1972). J. ‘vol. Biol. 72, 765-777.
432
J. I,.
MA4SJ,EY
Oberg, B., Saborio, J., Persson, T., Everitt, E. & Philipson, L. (1975). ./. l’irol. 15, 1X-2()7. O’Farrell, P. (1975). J. Biol. Chem. 250, 4007p4021. Pato, M. & von Meyenburg, K. (1970). Cold Spring Harbor Syrnlj. Q~uant. Biol. 35. 497-.i~4. Richardson, .J., Grimley, C. & Lowery. C. (1975). Proc. A’at. Acad. A’ci.. U.S.A. 72. 1725-1728. Scherberg, N. & Weiss, S. (1972). Z-‘roc. ,Vat. .4cad. Sci., li.8.A. 69, 1114 111X. Silverstone, A., =2rditti. R. & Magasanik, 1%. (1970). Proc. Xat. .-lead. Sri., iI.S.A. 66, 773-779. Steers, IX., ,Jr, Craven, G., Anfinsen, C. & Bethurle, J. (1965). J. Viol. Chew. 240, 2478-2484. Ullmann, A. & Perrin, D. (1970). In The Lactose Operon (Beckwith, J. & Zipser, I>., ~1s). pp. 143%172, Cold Spring Harbor Laboratory, Cold Spring Harbor. New York. Wallenfels, K. & Golkcr, C. (1966). Biockem. Z. 346, 1 12. Wallenfels, K., Sund, H. S: Weber, K. (1963). Biochem. 2. 338, 714 727. Wileox, M. (1971). In ,%fetaboZic Pathways (Vogel, H., ed.), l-01. 5, pp. 143~ 172, Academic Press. New York. Wilson, ,J. (1973). J. Afol. Biol. 74, 75S757. Zabin, 1. & Powler, A. (1970). In The Lactose Operorb (Beckwith. ,J. & Zipsor, D., ~1s). Cold Spring Harbor, K\‘rw York. pp. 27.-48, Cold Spring Harbor Laboratory, Zipsor, D. (1963). J. 12101. Biol. 7, 739-751. Zipser, D. & Perrin, D. (1963). Cold Sprin.g Harbor Symp. f&ant. Biol. 28, 533-538. Zipser, D., Zabell, S., Rothman. ,J., Grodzicker, T., Wenk, M. & Novitski, M. (1970). J. Mol. Biol. 49, 251-254. Zubay, C. (1973). Anwc. Rev. Uenet. 7, 267. 288. Zubay, U.. Chambers, D. dt Cheong, L. (1970). 111 The Lactose Operwr (Beckwith, ,I. & Zipser, D., cds), pp. 375-392, Cold Spring Harbor Laboratory, Cold Spring Harbor, Ken, T;ork.
(1978) 125, 407-G%
Synthesis and Degradation of Termination and Premature-termination Fragments of /?-Galactosidase in Vitro and in Vivo
(Received 13 September 1977, a,td in revid
,fornt
4 July
1978)
A rrumbcr
of abnormal polypept,ides whicl~ HI‘ a11 t,arly amber mutation in the 2 gene, is degraded iI1 ttle in vitro system. The rnwhanism of degradation appears t,o be specific for small abnormal polypeptides. 1nttwral wmitiation polyprptides generated by nonsense mut,ations, which have hwrl found in viva. are not detrct,ed irl thrl i?, vitro protein synthesis system urrdrr t,lrv conditions 71~4 IIerc. /Cscherich~ia
1. Introduction Mher mutzitions or mistakes which occur during transcription or translation oan lead to the synthesis of abnormal polypeptides. Polypeptides resulting from mutations can be of several types. Nonsense mutations produce protein fragments terminating at bhe altered codon. In addition. certain nonsense mutations result in the synthesis of int’ernal reinitiation polypeptides. due to initiation at a site downstream from the nonsense mut’ations and termination at the normal t)ermination site. DeleCon mutants can give rise to a number of types of fragment. Internal deletions of some multiple of t’hrre nucleoCdes (in-phase deletions) produce fragments with normal ends, but with amitm acids deleted internally. Internal deletions of a non-triplet number of nucleoticks result in bermination at an out,-of-phase triplet, and in some cases also generate
;Q 1978 Academic
Press Inc. (London)
Ltd.
408
.I. 1.. \l=\SLKY
internal reinitiation fragments. Prematurr-trrmillirtion fragments aIso t)r earlv quit,ters art’ readily seen in the products of protein svnthesix in vitro (Atkins &, (A~steland. 197.5: Chambers & Manley, 1973: Dottin rt al., 1975: Kerr et al.. 1972: At’kins et al.. 197.5). The view has been that this is an arbifa,ct of the in Gtro systems. However, if premature termination does occur frequently in viva. it might not IW apparent for at least two reasons. First, abnormal polypeptides. including prematurcbt,ermination fragments which result from nonsense mutations, are trrquently rapidly and specifically degraded in viwo (for review . see Goldberg & Dicta. 1974). Nothing is known about the mechanism(s) by which these polypeptides are recognized and then degraded, but several mutants deficient in degra,dation in ciao have been isolat,ed (Bukhari & Zipser, 1973 ; Apte et ul.. 197.5). This t’ypc of specific degradation has not lxw~ demonstrated ire Second, such premature-termination fragments would be likely to be difficult to det,ect hy standard biochemical t,cxchniqut:s. It is likely that these molecules would he lost, by routine purification schemes. However, w&in techniques might be expected to uncover such polypeptidrs, if they exist. ITor example. a,ntibody precipitation followed by sodium dodecyl s~~lfatc?/pot~~~~cr~lamide gel elect’rophoresis of the precipitated proteins should revt~al any ~~~~c~~i~~t~n~~f~-tarrnillntion fragments which contain exposed ant,igenic drt,erminant,s. in this paper 1 report the charactrrizatiori of sc:vt>ral types of abnorma t polypeptides synthesized CAL and in ‘ciao. The potypeptides synthesized from the % gene of the lac operon of Escherichia coli. which encodes ,B-galactosidase. have been purified by antibody precipitation and analgzed on SDS~/polyacrplamide gels. 1 describe a set of specific protein fragments. and provide evidence that they are dutt to premature t,ermination of protein synthesis within thtt Z gene. These fragment,s art’ vitro. The total amount of these fragments is estimated to made in. viva as well as hr at least 23O!;, of the t,otal B gent’ protein. These results show that, premat,uretermination, at least, in the case of /%galactosidasc, is not an artifact of protein synthesis i?z vitro, and suggest that synthesis of aberrant polypeptidrs may he much more common than previously thought. Fragments synthesized vitro from DNAs containing amber mutations have also been examined. They are identica,l in size to the corresponding fragments made in l!ivo. However, unlike many nonsense fragments synthesized in 1~it10~ which are rapidly degraded, the fragments synthesized in vitro. with one exception. arc very Aable. The exception, a fragment produced by an early amber mutation, cannot be detected k)y SDS-containing gel electrophoresis, and evidence is presented that, the IMP TLOUO synthesized mutant polypeptide is specifically degraded itb vitro. (‘il,ll
vitro.
vitro
it,
it1
2. Materials and Methods (a) Materials Rabbit arlt~i-8-galactosidase autisenlm UXR a gift from 31. Zipser. It, was pro-adsorbed \yith an equal voh~rnc of a cortcentratcti ext-ract made frown a lac deletio!l st.rain (strain 1 in ‘Fable 1) by incubation in the cold for 3 to 4 h. In one case t,he i~rl~nllno~lobulin fraction was purified as described by Atkins & Gest,elanp (1975). [35S]methionme (200 to 400 Ci/mmol) was either from New England Nuclear. Bost,on, Maas. or made as drscribed by t Abbreviations used: SDS, sodium dodecyl sulfate; CAY, cat,abolite act,ivator protein
TII-Tris (pH 7,J-). 200 IIIRI1 RI-urea, and 1 l/o Tritorl X100). or, in some earlier c~xperirnor~ts (tlrc KCl, 1 mrvr-MgCl,, results of one of these are sl1own ill Pig. 5), with 1 ml of buffer 13, wliiell is k)llffiar CTxvit~llolct. tho urea and Triton X100. The addition of I >I-urea plus 1 “() ‘I’ritoti S IO0 to t I)(’ ar~t~rl)oti~~ precipitation buffer, a modification of a tt~chniquc~ originally drscribrd t,y Oberg et a/. (1975), was extremely effective in reducitlg background noll-specific l”.f.‘,il.)itatiotl. I IIsoluble material was removed by centrif1~gat,ion at 30,000 g fi~r 15 IIlirl. (f) Protein
synthesis
in vi\-0
Cells were grown in 10 ml MQ medium (Miller, 1972), wit11 glycerol as tile carbon source. at 34°C. Any required ammo acids (see Table 1) were u,ddcd at, a concn of 50 &ml. ‘lYLc cells were grown until they reached a Klett reading of -85. at which t,imc 5 x 10F4 &Iisopropyl-thio-/3-D-galactoside \\-a~ added t,o arly cultures tlot const itutivr for tllrx (1xpression of the Zac operon. One minute later. 100 &i of 135S]Inet’lliorline w(‘re added and the cells were allowed to grow for 3 more min, except where noted. They were then harvested on ice, washed with 2 ml buffer H, and rcslispended in I ml huffkr C. The cells were lysed by sonication in an ice-bath for 30 s wit,h a Hiosonic IV sonicator. Debris was removed i,v centrjfugation at 30,000 g for 15 min. Roughly BOY/, of thra added radioactivit~y was recovered as trichloroacetic acid-insoluble polypeptides. (g) Analysis
of radioactive
1acZ gene-specijicd
polypeptides
Samples were routinely analyzed by precipit,ation with anti-p-galactosidaso antiserum, followed by SDS/polyacrylamide gel electrophoresis. This was done in or~e of two ways. For all the in vitro samples, and those in viva samples not, constitutiva for lac operon expression, approx. 1.5 pg of unlabeled fi-galactosidase was added to the 30,000 g supernatant fraction, to serve as carrier for the antibody precipitation. (TIE carrier was added as a crude extract, prepared from strain 3 in Table I, essentially as described in the preceding section except that no label was added.) A sufficient amount of antiserum was added to precipitate all the carrier, and reaction mixtures were incubated at 0°C for a minimum of 3 h, but usually overnight. The amourlt of /3-galactosidase-related polypeptides in the labeled extracts (in pg), is so small that, it does not affect the antibody titratioll. No differences were ever observed when precipitation was performed for different, lengths of time within this range. Three samples were prepared from strains constitutivr for expression of the lac operon (strains 12 to 14 it1 Table I ). In tile in viz10 samples prepared from these strains, the amount of /3-galactosidase-related polypeptides may not, be insamples. significant compared to tile 1.5 pg of carrier, a.s was the case with t,he prcviolls In the case of strain 12, which is wild-type, the concentration of ,6-galactosidasc can be determined by enzyme assay. A volume of sample which corresponds to 1.5 fig of 8. galactosidase was removed from bhe 1 ml of supernat,ant described in the preceding section (this is roughly 50 pl), diluted to I ml with buffer C, and precipitated directly
TERMINATION
FRAGMEKTS
OF p-GALACTOSIDBSE
411
with antiserum. For strains 13 and 14, I determined in preliminary experiments how much antiserum was necessary to precipitate l-5 rg of carrier in the presence of an arbitrary amount (100 ~1) of each of the 2 mutant extracts. This amount of antiserum was then used to precipitate 1.5 rg of carrier plus 100 ~1 of each labeled extract, in 1 ml of buffer C. In all the samples, precipitates were collected by centrifugation at 20,000 g for 10 min, washed 2 x with buffer C, and dissolved in 50 ~1 of sample buffer described by Anderson et al. (1973) (0.08 M-Tris (pH o-8), 0.1 M-dithiothreitol, 2% SDS, 10% glycerol, 0.004”6 bromophenol blue). The samples were boiled for 2 to 5 mm, and a sample (5 to 25 ~1, depending on the sample and the experiment) was loaded on a loo/ SDS/polyacrylamide gel, which was made, and run, stained and destained according t,o Anderson et al. (1973). At this point the samples could be compared to determine if the efficiency of antibody precipitation was the same in all cases, by comparing the intensity of t,he stained flgalactosidase carrier bands. Part of a typical stained gel is shown in Fig. 2(b). Gels were then dried under vacuum and exposed to Kodak X-ray film SB-54, usually for about 1 to 3 days. When quantitation of band intensity was required, autoradiograms were scanned with a Joyce-Loebl Chromoscan. and the peaks were integrated with the aid of a Numonics planimeter. (11) Auto- a complemenlation
assay
Auto-cc assays were performed as described by Morrison & Zipser (1970), except that in all cases the auto-a donor was a loo-p1 protein synthesis reaction mixture diluted to 200 ~1 with buffer D (0.02 M-Tris (pH 7.2), 0.2 M-p-mercaptoethanol, 0.04 M-EDTA, 0.01 XNaCl).
3. Results (a) Comparison
of in vivo and in vitro synthesized polypeptides
of several mutations which result in the production of polypeptide fragments in vivo are shown. These fragments lack enzymatic activity, are of lower molecular weight than the wild-type protein, and are degraded in vivo with half-lives as short as five minutes (Goldschmidt, 1970a; Bukhari. 1975; Manley, unpublished results). To study the synthesis of the fragments in vitro, these various mutations were transferred to Xpluc5 transducing phage by B. Apte & A. I. Bukhari, who generously provided them for these experiments. The phage DNA was used to direct the synthesis of proteins in vitro, as described in Materials and Methods. Z gene-related polypeptides were purified by antibody precipitation and analyzed by electrophoresis in SDS/polpacrylamide gels (see Materials and Methods). Figure 2 compares in vitro synthesized polypeptides and the corresponding in viuo Figure
1 is a map of the Z gene of the lac operon of E. coli. The positions
Fro. 1. Genetic map of the ZacZ gene. The numbers above the line represent amber mutants. x( 1) is a mutation which results in the synthesis of an internal reinitiation polypeptide in the double mutant 646rr( 1). The broken lines define the 3 complementation regions of the gene. The 2 enclosed areas underneath the line indicate deletions.
41”
.I . 1,. JI .\ s Lb: \
products. The mutant fragments synthesized ir/ vilro and it1 viuo wmigrate, providing strong evidence that the mut,ant pol,ypept,idcs produced it/ vitro aw t hc si~~~l(~;IH t host. synthesized in vivo. As equal amounts of each of the itl ~ifro samples UWY: loaded onto t,tic gel, the nnAar yield of the various % gene-derived polypeptides can be estimated. 1Cor cxamplr; the introduction of IO’-5. a mutation which eliminat,es the requirements for the catabolic~ activator prot,ein and cAMP for lac optwn transcript,ion (Silverst~onc it ccl.. 1970). into ApZac5 resuhs in a great increase in t,hc amount of /3-galactosidase synt,hesized it/ r!itrct (Big. 2 slots d and e). (The lvsogen from which this DXd \vas prepared \\w kindI> provided by P. Bazzicalupo.) Also Xplr~6 IT\:-5 DNA tlireot~s t,he synt,hesis of approximately ten times as much enzyme as does hpZnc.5 DXd (Eron & Block: 1971 ; set: also Materials and Methods). Thus, even though the S-30 extract used in t,hese experiments is CAP+, it, seems that activcb CAP protein is a limiting factor under t~hew conditions, which are optimal for a high yield of P-gaI~bct,osid~~s~. A comparison of ttw intensity of tha wild-type potypeptidc hand lwoduwtl t)y hpZac.5 DE\;;{ wit,h ttlc int)txnsities of the bands produced in vitro t)y tllcl various mutant, Dl\‘As sl~ows tllat’ iill tIlta as \\,($I1 i18 tmllc liolypel~tidc~ s,vnthesiz,cd nonsensf: mutant termination polypeptitlcs. from DNA containing the small in-phase dt~letion. A21 _ art present in at lcast 75’1, ttic protein yield. assuming an equal distribution of’ mcttrionim~ wsiduw throughout (Fowler $ &bin, 1977). This is in strwli cwnt,rast, t,o ttle yield 0litaincYl with tllc samf fragments synthesized it/ tlivrt. The nonsw~s~~ tc~rmination tiagmc~nts a~‘(’ recovc~retl in yields of at best, .W’;, of tlrc wild-t,ype lwc*l. we11 aftw ii pulse label. Other mutant, polypeptides that, one might, expect, to tint1 synthesized it/ citro are r~:c:ovwcd in veq low yields, if at all. The d&l fragment. \\ hich most prohalily results from an inphaw internal deletion, is recovered in very poor aii(l variable yield. TWO internal reinitiation polypeptides which can he detected it/ ri?vo. thaw from the mutat1t.s 2 t 77 (RS in Pig. 2) a,nd 54$7(l) (Zipwr ?‘I al.. IWO: Xpttb ct ~1.. 1975) (data for .‘,-l:?n( 1) not sho\vn), cannot, be (let&cd at all iI/ vitru under t,hcs wnditions usetl Ilcre. In addition, thtb 5457r( I ) nonsense termination fragment’s from t\\-0 carty % gvnc~ amtw mutations, (see Fig. 1) and @24(%v\-hichmaps M\vet~tl 54hr( I) and 2177). c:annot lie tletectt~d. This is true not only for the iti vitro products Ijut alsc~. ;\t~ lc,ast in that c*aso of’ 5457~(1). for the in vivo polypeptide (dat,a not sho~+n). l?inally, in addit,ion to the major component in c~ch sample. a number of ot’hrr usually bvith greater c,lcct~ophorc:tic mohilities. can tw tlet~ected in most polypeptides. of t,he samples.
(b) Lower wtolecdw
weight frqpetttn
What is the origin of these polypeptides. \\-ith apparent molecular weights less than unrelated to full-sized /3-galactosidase protomer? ‘I’ht?y could be, for example. /3-galactosidase and t,hus artifact,s of the antibody precipitation. The follo\ving rxperimerits show that at least nine are specific products of t,ht Z gene (indicated by solid lines in Pig. 2). slots a and 1) of Figure 2(a) are antibody precipitates of 135QImethiotiirie-labr.1~~1 (sxtracts. from both ill vitro and it), vivo. in uhich no Znc DNA sequences were present. None of the polypeptides present in t,hc sampk. w thatf do contain lac DlVA can be detected. (It should be noted that the amount of physical material precipitated is tht same in all cases, owing to the addition of a large molar excess of unlabeled wild-type b-galactosidase; see Materials and Methods.) But perhaps aggregation which results
a
bc
defah
i
ik
Imn
BG
7 0 9
-H
(a)
414
J. L.
MANLEY
in non-specific precipitation occurs before the addition of the unlabeled wild-type was checked in two ways. The carrier. Thus the specificity of antibody precipitation first shows that non-specific aggregation during preparation of the crude extracts (e.g. during sonication) does not occur at a level detectable by the subsequent’ antibody precipitation. Strains 11 and 12, which are isogenic lac deletion strains except that one contains a X prophage and the other a XpEac5 prophage. were grown as described in Materials and Methods. The cultures were divided in two, and half of each wa,s labeled with [35S]methionine for three minutes. Then a large excess of non-radioactive methionine was added to stop incorporation of label. Each labeled culture was then mixed with the unlabeled reciprocal culture, chilled on ice and processed as described in Materials and Methods. The results of the antibody precipitation are shown in Figure 3 (slots a and b). There are a number of specific polypeptides of lower molecular weight tha,n the full-size (135,000 M,) polypeptide in the sample prepared from the cells lysogenized with hpZuc5 (numbered 1 to 9), and these are absent in the sample from the cells containing the h phage. This argues that these polppept’ides do not result from artifactual aggregation during sample preparation. I next wished to determine whether synthesis of these fragments is regulated by the lac controlling elements, as would be expected if they are actually products of the Z gene. Strain no. 15, which contains a wild-type copy of the entire lac operon, and is therefore inducible for synthesis of the lac enzymes (Aplac5 lysogens do not contain a functional I gene and are therefore constitutive), was grown up in duplicate cultures. To one the gratuitous inducer of the lac operon. isopropyl-thio-/l-n-galactoside, was added when the cells reached a concentration of 5 x lOa/ml. Two minutes later, [s5S]methionine was added, followed five minutes la’ter with a large excess of unlabeled methionine. The cells were harvested after 30 minutes of additional growth. To the other cultures [YS]methionine was added first, followed five minutes later by the chase. One minute after this, isopropyl-thio$-n-galactoside was added. A sample was harvested a,fter two minutes, and the remainder after 35 minutes of additional growth. The samples were processed as described in Materials and Methods (carrier p-galactosidase was added to the sa,mple induced for only two minutes to equalize the total amount of enzyme in each of the three samples). The results are shown in Figure 3 (slots c. d and e). Some polypeptides are present in all three samples. However, a number of polypeptides are present only in the sample induced before addition of the label, and these therefore must’ be specific products of the 2 gene. A close comparison shows that the mobilities of t.hese polypeptides are the same as those of the proteins indicated by lines in Figure 3 (slot a). On the basis of these results, and other experiments discussed below, I conclude that the polypeptidex. numbered 1 to 9, are products of the 1acZ gene. The fragment indicated hp the broken line will be discussed below. The polypeptides numbered 4 and 5 are only occasionally resolved. For example in Figure 3 (slots a and e), the autoradiographic bands are not well resolved. whereas in Figure 2 they are. Note that slot e (Fig. 3) represents a 30-minute chase. I have consistently observed that there is no change in the intensity of any of the &c-specific bands during the course of such a chase. This result. which is consistent with in vitro results presented below, argues that the fragments do not arise by proteolytic cleavage during growth. Also, the fact that the fragments are produced in st,rain 15 shows that they are not a result, of t,he presence of t,hc h prophagc, which is present in the other ijr vi~io samples. The nine polypeptides indicated in Figurc 2 have the same mobilities as those in
7a-9-
FIG. 3. Autoradiograms of SDS/polyacryIamide gels which shoxv that p-galactosidase-derived fragments are not artifacts arising from the antibody precipitation reaction. Slots a to e, controls for specificity of antibody precipitation (see text for details). a, [35S]methionine-labeled polypep. tides precipitated with anti-/l-galactosidase serum from strain 12 (Inc+ ). b, [35S]methionine-labeled polypeptides precipitated from strain 11 (&UC). c, polypept,ides precipitated from strain 16, which had been labeled, chased, and induced for 30 min. d, same as c, except, that cells were induced for only 2 min. e, same as c, except that cells were induced before addit)ion of label and chase. The solid lines indicate &galactosidase-specific fragments. The broken line indicates a fragment,, also apparently /?-galactosldase-specific, which appears to be missing amino acids from the ammoterminal end rather than from the carboxy-terminal end like thr other fragments (see text,). f to g, [35SJmethionine-labeled polypeptides synthesized in vitro and analyzed without antiserum purifioation. Immediately after the DNase step (see Materials and Methods), 1 ml of ice-cold 80% acetone was added to each sample. The precipitated protein was collected 10 min later by centrifugation, dissolved in 100 ~1 of gel sample buffer, and heated t,o 100°C for 2 to 5 min. A tot,al of 5 ~1 of each sample was then loaded on an SDS-containing gel. The templates were: f, X DN.4; g, hpZac6 UV-5 DNA. Lines indicate detectable fi-galactosidase fragments.
Il(i
.I. I,. 11:\ s 1,13Y
Pigurc: 3 and are present in both the i//, V;WJ and j/l ~?ifr~ sillrlplt’s. Ho\~c~vt~~~.i\ ~l~ltr~l)t~r of ~loll-spe~iti~ polypeptidrs t,hat, vary in I)otlr amount iit>(l tlurrll)c~r iI1 t hc* (lifY(*r(tnt samples il1x-L also apparent. lqor ~~xampk~. ill I~iyurc~ ;I (slot a) tlrcw iiu~ virtikally 110 non-specific polypeptides. but in t,lrcssamples in l~‘igurc~ 2 tllt’l’(’ ill’ the following results. The kinetics with \vhich th(a fragments appear. as assayed on SDS-containing gels, can provide some information as t)o how they arc fortncd. For example, in the case of wild-type /3-galactosidase, if the final step in the formation of the fragments is the same as it is for t,hc full-size prot,omer. i.c. tc~rrnination of th(b polypeptide chain, then one would expect to SW t,hcs c*oncc~ntration of t hc fragments increasing at the same rate as the concentration of the 135.000 LIJ~protomor. Howcvrr. if Ohere is an additional cleavage step in thr formation of t,htl fragmtbnts. t)hcn out might expect the concentration of bhe fragment’s to incrcbase only after a lag. when cornpared to the complete polypeptide. Figurt, 1 sholvs the products of protein synthesis in vitro directed to hpZac5 1’\‘-.5 DNA. for various timrs of synthesis ranging from five to 50 minutes. The P-galacl-osidasc~-~~l~lt(~(~ fmgrncnts (solid lines) do not sho\v a lag in appearance compared to \vild-type /?-ga,lactosidase. I II fact. itt bll(* (xarlip::t t’ime point at which t,he 135.000 ;I/,. pol?peptidc (*a111~ dt%rct,tbd (7.5 tnin) many of the smaller polypeptides cau also I)t: detc~ct~ed. at roughly t,hta sam(’ intensity ah thcb wild-type. However, at the conclusion of the reaction t,he intensity of the band produced by the full-size polypeptide is of the order of tcu times greatjcr than that of any of the fragment bands (see also Table Z), so it appears that the fragmc>nts are synthesized at! a greater rate. compared to t hc full-size protein. early in t’hcl reaction. \;c’h~ this is so is not, clear, but these kinetics of appearance provide strong c~vidcncc~that the fragments do riot arise by proteolytic clraragc of filll-sized ,Qalac*tosidast~. Thrrt: is t)j, one fragment, however. that’ appears with kinetic s consistent with it. originating degradation of a larger polypeptide. This fragment is shown hy a Ijrok(bn lint, in Bigurt~ 4, and is not, one of the polypeptides indicated it1 Figures 2 and 3. Another way to ask whet’hcr or not. th(b fragments ar(~ genrrattad I)y post-translational cleavage is to see if fragments continutb to hc generated afttxr protciu synthesis is halted. In the experiment sho\z,tr in Figure 5. various Xplac5 DNA wart’ used to direct protein synthesis in the in vitro sy&:m for 20 minutes. Than ~~llorwlnpht,l1icol
ii
(69)
R!) (30)
70 (61)
15 (36)
1.5
I .!I
3.1,
i.1,
I.4
I.li
I.0
4.0
s.9
6.6
13 16
1 .!I
4.7
x.2
ti.ti
2.4
I .:
. (1972). J. ‘vol. Biol. 72, 765-777.
432
J. I,.
MA4SJ,EY
Oberg, B., Saborio, J., Persson, T., Everitt, E. & Philipson, L. (1975). ./. l’irol. 15, 1X-2()7. O’Farrell, P. (1975). J. Biol. Chem. 250, 4007p4021. Pato, M. & von Meyenburg, K. (1970). Cold Spring Harbor Syrnlj. Q~uant. Biol. 35. 497-.i~4. Richardson, .J., Grimley, C. & Lowery. C. (1975). Proc. A’at. Acad. A’ci.. U.S.A. 72. 1725-1728. Scherberg, N. & Weiss, S. (1972). Z-‘roc. ,Vat. .4cad. Sci., li.8.A. 69, 1114 111X. Silverstone, A., =2rditti. R. & Magasanik, 1%. (1970). Proc. Xat. .-lead. Sri., iI.S.A. 66, 773-779. Steers, IX., ,Jr, Craven, G., Anfinsen, C. & Bethurle, J. (1965). J. Viol. Chew. 240, 2478-2484. Ullmann, A. & Perrin, D. (1970). In The Lactose Operon (Beckwith, J. & Zipser, I>., ~1s). pp. 143%172, Cold Spring Harbor Laboratory, Cold Spring Harbor. New York. Wallenfels, K. & Golkcr, C. (1966). Biockem. Z. 346, 1 12. Wallenfels, K., Sund, H. S: Weber, K. (1963). Biochem. 2. 338, 714 727. Wileox, M. (1971). In ,%fetaboZic Pathways (Vogel, H., ed.), l-01. 5, pp. 143~ 172, Academic Press. New York. Wilson, ,J. (1973). J. Afol. Biol. 74, 75S757. Zabin, 1. & Powler, A. (1970). In The Lactose Operorb (Beckwith. ,J. & Zipsor, D., ~1s). Cold Spring Harbor, K\‘rw York. pp. 27.-48, Cold Spring Harbor Laboratory, Zipsor, D. (1963). J. 12101. Biol. 7, 739-751. Zipser, D. & Perrin, D. (1963). Cold Sprin.g Harbor Symp. f&ant. Biol. 28, 533-538. Zipser, D., Zabell, S., Rothman. ,J., Grodzicker, T., Wenk, M. & Novitski, M. (1970). J. Mol. Biol. 49, 251-254. Zubay, C. (1973). Anwc. Rev. Uenet. 7, 267. 288. Zubay, U.. Chambers, D. dt Cheong, L. (1970). 111 The Lactose Operwr (Beckwith, ,I. & Zipser, D., cds), pp. 375-392, Cold Spring Harbor Laboratory, Cold Spring Harbor, Ken, T;ork.
