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Use of gene fusions to isolate promoter mutants in the transfer RNA gene tyrT of Escherichia coli
./. Mol. Biol.
(1979) 130, 303-315
Use of Gene Fusions to Isolate Promoter Mutants in the Transfer RNA Gene tyrT of Escherichia coli MICHAELLBERMANJ~AND
ijepartment
.JON BECKWITII
of Microhioloyy and Molecular Harvard Me&d School Boston, Mass. 02115, U.S.A.
Genetics
(Received 30 October 1978) LIL tllcl prwed~ng paper. we tlmcribt~ tjtlc isolatiorl of stmins ill whicll tile lac genes are fItsed to and under the control of the structural gene for tyrosine tRNA, t!/r!Z’. Using a wioction for mutants wllictl have lowered expression of the lac genes in sue11 fusion strains, we ha\re detrctrd derivatives whictl carry mutations in ttw t?yrT promoter. These mutations result in a reduction in expression of tile lac genes irl the tyrT-Zac fusion strain to a few prrcent, of tlrr parent levels. These mutations vc~ldtl tw genetically recombirlcd orlto a copy of ttle tyrT germ \v-llich carries ali amber suppwssor allele. Amber sl>ppressor dorivat,ivns wtiictl carry these promotw mutations are greatly reduced in tlloir ability to suppress amber tntltat,ions. consist,ent, with a reduction in transcript,ion of the tyrT gent. Based upon thei int,o three effects on oxpression of the tyrl’ getw, the mutants could be divided clnsscs. Thrw are tho first promoter Instants irl a st,abla RNA gene.
1. Introduction In the prectding paper (Berman & Beckwith, 1979) we showed that it is possible to fuse genetically the transcription controlling sites of a transfer RNA gene, tyrTS> to the DXA sequences which code for the enzymes of the lactose operon (lac). The expression of the structural gene for ,%galactosidase (la&) by the fused hybrid operon is cont,rolled co-ordinately with stable RNB synthesis in Escherichia co&. Insertion tnutat,ions which were isolated as inactivating the tyrT gene were shown to inactivate ctxpression of tjhe Zac genes from the fused operons. In a similar genetic approach WC now show that mutations which are isolated as inactivating expression of a fused operon will, following the appropriate genetic crosses, affect expression of the tyrT Ktme. .4lt,hough numerous mutations within the tyrT gene have been isolated, no mutations affecting tht regulation of this stable RNA gene are known (Smith, 1976). I’sing a fused tyrT-lxx operon for genetic studies, we have SCated studies to select t To whom correspondence should be sent at : Section of Microbiology I )Ivision of Biology and Medicine, Brown TJniversity, Providence, R.I.
$ The genetic
abbreviation3
used arc according
to Rachmann
and Molecular 02912, U.S.A.
et al. (1976). lyrT
Biology.
is t,he gcwt*
coding for the minor species of tyrosine tRNA. The amber suppressor allele of this gene was used throughout,. This allele has been called Ru,,,, su3 as well as sup?’ previously. A bact,eriophagc: carrying this gene (@Opsu&) has bern isolat~c~d previously (Russell et rrl., 1970). Su’ indicates suppressor phenotype, Su” non-suppressor phenotype, and Su * weak suppressor phenotypta. XG, .5-t~romo-4-chl~~~~~-~~-i~~~~~lyl-~-n-gala~~~~si~~~: tONPG, 0.nitrophenyl-p-D-t,hiogalactoside. 303 0(,1’2--“8313/79/150303-
13 $02.00/O
0
1978 Acaclcrnic
I’ress Inc. (London)
Ltd.
304
31. I,. .BERMAN
ANI)
.J. HECKWITH
mutations within the tyrT gene and possibly in genes for other factors which control the t.ranscription of the tRNA gene. Gene fusions to the tat operon have been used previously to obtain mutants affecting the expression or the regulation of hybrid fusion operons (DBbarbouillB et al., 1978; Campbell et al., 1978; Reznikoff & Thorntou. 1972). Here we report the isolation of promoter defective mutants of the tyrT gent’.
2. Materials and Methods strains (a) Hacterial l?usion strain 6 was constructed as described irt the preceding paper. This strain carries a fusion of the lac.2 and ZacY structural penes t,o the controlling region of the tyrT gene. The mutants described in the present study were all isolated from this fusion strain. The parent of this fusion strain was derived from strain M7017 from the collection of J. Beckwith. The genotype of strain M7017 is: F-~ucZ,,,~,,. trpnm3 (A’). SIP. relA. Ot,her bacterial strains have betn described in the preceding paper.
The genetic techniques and t,ht, assay of /3-palactosidase have been described ceding paper and by Miller (1972). The cl>-c, 5-l~ro~no-4-c~~loro-3-indolyl-~-~-~alactoside a substrate for /3-palactosidase, the product of t,he lacZ gene. The enzyme cleaves less compound to produce the blue dye indigo. TIIP production of 8.galactosidase strains correlates well with the degree of blne ~olr)r of colonies on minimal plates 40 pg of this dye/ml. (c) o-Nitroph.erc.yl-8-D-t//ioyalactoside
in the prcis this colorby various containing
selectiorc
Spontaneous mutants resistant, t)o tONP(: \VCLI‘Vselectjet 011 succirlate (0.20,,) minimal plates containing lo-” $1.tONPG and 2Opg X(:/~ul. ‘I’lle pritlciple of tllis selection is described in Kesrllts, section (a). Tllr selection was couductjed a.t 30°C for 3 to 5 days. The level of expression of t.hr lac genes in fusion strain 6 is hiyll enough to inhibit the growth of this strain on this selective medium. Survivors appeared at a frequency of 2 to 3 x 1V7. The selection is based on t,hr method of Hopkius (1974). (d)
1’4
amber
p//aye
test
the ability of strains to support t,lrct Suppressor plienot’ypc* can be tested by ohsen-ing ggowth of T4 phage amber mutants. A fresh Hershey agar plate is overlaid wit,h a lawn of the strain t.o be test,edusing Hershey top agar (Biswas nt Gorini, 1972). Appropriate dilutions of the phage lysates are applied in patclles wit11 tile aid of paper st,rips as described pre1965). In all test,s a wild-type T4 phage is included as well viously (Brenner &z Beckwith, T4 mutants wer<> as control plates seeded with an Su” and an Su+ strain. Pour different obtained from the collections of L. Gorini and H. Wanner and proved to be the most useful for this analysis (Table 1). These phages were used at. a concentration of about lo5 plaqu+ forming units/ml. Weak suppressor strains support varying amounts of lysis when comT4D phage. pared with Su + strains. Su” strains will only allow growth of the wild-type
(H) l,&atior/.
of tra.nsduciny
phage carr,ying
the weuk mppressor
allelex
described in this study were Specialized transducing phages currying t’ht? Inutations isolated as recombinants with the plaque-forming ptlage @OpsuG, (Russell et al., 1970). This phage which carries the amber suppressor allele of tyrT was kindly provided by .J. Smith. As described in Results, section (d) (see Fig. 1) transducing phages carrying the presumptive> promoter mutations are obt.ained in Iysates cont,aining the parental phage as well. The by allowing plaques to suppressor activity of transducing phages can he monitored form on la,ctose indicator plates seeded witlk a strain carrying a lactose amber mutation. 1972) allows a distinction t.o be made bcThe use of tet,razoliurn indicator plates (Miller. tween a phage carrying a weak suppressor allele and a phage carrying t,he wild-type sup) is uscld as an indicator strain on fresh plates, pressor allele. When strain M7017(l~cZ,,~~*
tRNA
PROMOTER TABLE
305
MUTANTS
1
T4 phage ckmIlent
Phagr
-
~,,r dgbtails SW Mat,&&
T4L)
Wild type,
T4 amli
(‘atalytic
requirctment
T4‘amH39
(‘atalyt,ic
requirement
T4 amNl33
Stoichiometric
requirement
T4 amN3 19
Stoichiometric
requirement
and Met~hotls, sert,ion ((I).
ptiage produce red plaques, while phage carrying the Su” allele producr St1+ t,ransdlxing \\hite plaques, and Su * (weak) transducing phage produce light red plaques. The confluent llac- laun is whit<>, which is the reverse of the usual color reaction on this medium. Ptlages carrying weak suppressor alleles were used to lysogenize strain M7017 in t,trc, following way. Since @Opsu,,,+ has a defective attachment site, this bacteriophage lysogerlizes host cells ver>. poorly. In order to select stable lysogens, LO3 cells were infected at R Innltiplicit,y of infection of 0.1 and drown to saturation in I, broth (Miller. 1972) ox-ernightj. Lysope~ls were isolated from the difl’erent cultures by st,reaking the cells 011 lactose MarConkey indicator plates in the presence of @Oc22, a clear mutant of 480. Only cells irmnrrfw to 480, i.0. lysopens, will form colonies on these plates. Lysogens of the phages indicator plates, yet appear carrying the weak suppressor alleles are Lac + on MacConkey Lac - on the less sensitive tetrazolium indicator medium. These lysogens also grow poorl? on minimal plates in the absence of added tryptophan (M7017 is trpam). In contrast. lyso~enq of #W~SII,+,, are fully Lac’ and Trp + on all media tarsted.
3. Results (a) Selection of Lac - ,mutants of &ion.
strain 6
In the preceding paper we described the construction and properties of strains carrying fusions between the tyrT gene and the structural genes of the lac operon. lTsing selective techniques based upon the expression of the lac enzymes, it should be possible t,o isolate from a fusion strain Lac- mutants which are due to defects in the regulation of the tyrT gene. Such mutations could be within the site controlling transcription initiation of the fusion (i.e. within the tyrT gene regulatory sequences) or mutations in other genes required for tyrT expression. Among mut’ations linked to the tyrT-lac fusion, insertions, deletions, and other chromosomal rearrangements disrupting transcription from the tyrT promoter could be selected. It should be possible to recombine such mutations onto a wild-type copy of the tyrT gene and reduce its activity. Small deletions or point mutations within the RNA polymerase binding site would be ext’remely useful in defining the funct,ions of this region of the tyrT gene. We chose a procedure which has been used previously to select, mutants of the wild-type lac operon (Hopkins, 1974). hmong these mutants of the Zac operon, a number have been shown by DNA sequence analysis to be point mutations in the promoter site (Dickson et al., 1975,1977). One of the tyrT-Zac fusion strains described previously was used to select similar mutants of the tyrT gene regulatory region.
3Ot-i
M.
L.
BERMAN
ANI)
.I.
13ECRWT’I’H
The selection t’echnique used is based on the fact that, Lac + strains arr senxitivt~ to the t’oxic lactose analogue O-nitrophenyl-p-n-thiogalactosida (Hopkins, 1974). This compound is transported by the lactose permease. the product of t#he second gene of the Zac operon (lacy). Since the ZacY gene product is required for transport, of t,ONPG int)o the cell, strains wit,h lowered levels of the permease will bc resistant to this cornpound. Among such tnutant’s are certain pleiot.ropir classes which have reduced levels of /?-galact’osidase as well as lact,ose permeasr. Among such mutants in t,he \\ild-t?;pcs Znc operon were mutant)s in the promoter site (Hopkins, 1974). Using this selective technique, we obtained survivors and screened t,hese co1onic.s for reduced expression of t,hc 1acZ gene in th(A following way. Fusion strain 6. a tyrT-Zac + fusion strain. produces up to 500/, of t,hc levels of Zac enzymes compared to the wild-type Zuc operon, and is sensitive to tONPG. From this strain. sponbaneous mut’ant’s resistant t,o tONPG were selected on plates containing the indicator dye X’G (see Materials and Methods. se&ion (c)). This indicator dye yields a blue color when cleaved by /?-galactosidase, making it. possible to screen survivors for enzyme production based upon t,he color of individual colonies. Spontaneous survivors appeared at8 a frequency of 2 to 3 >( lW1. Mutant strains which gave rise to light’ blue colonies. indicating leaky expression of the ZacZ gene, were purified from a number of independent, cultures of isogenic ,wZ.~+ ant1 YPZJ- clerivat(ives of fusion strain li (Table 2)t. !~ABLE 2
tONPG-rehstant
Spontaneous survivors tluction direcbly gene (see test).
from on the selective
mutan.t.s qf tyrT-lac
20 indrpendent, metlium. “Light
sfusion G
vultuwa wtm~ scrrenctt for /3-galactosidase pry,rcducetl cqwession of the ZacZ blw ” indicates
As expected, the majority of the survivors of exposure to tONPG were either Zac 1’ mutants, Lac+ tONPG resistant. or ZacZ mutants polar upon the expression of the distal ZacY gene. The first two classes of mutants form dark blue colonies on the selective plates in the presence of XG. These strains can be shown to produce the same levels of fi-galactosidase as the parent fusion strain (Table 3A). Generally, the third class of mutants form white colonies on XG plates. A number of these mutants proved to be amber, ochre, or UGA mutations in la& (data not shown). However, certain polar mutants in la& might retain adequate levels of fl-galactosidase production to form light blue colonies on the selective medium used. These mutants have the same t Mutations in the rel allele are known to affect stablo RNA gene control under a number ot culture conditions (Ceshel, 1975). Since this selection involves growth on selective plates containWOPC used to avoid any contribution of ing a poor carbon source, both reZA+ and reZ.4 - &ttins the state of the reZAgene to t,he selwtion.
tRNS
PROMOTER
so7
MUTANTS
TABLE 3 p-Galactosidase
levels of tONPG-resistant
mutants
/I-Galactosidase
Strain A. Fusion Fusion Fusion
6, ml+ /relA 6, lmY 6, lot + , tONPG’
B. Fusion Fusion Fusion Fusion Fusion Fusion Fusion Fusion Fusion
6, 6, 6, 6, 6, 6, 6, 6, 6,
ml+, ml+, T&I,
Zue-74 Zuc-119 Zac -20
relA, TelA, relA, relA, relA , relA,
h-27 lnc-45 lnc-51
h-9-6 Zac- 1 l-4 h-20-3
activity
536 480 551
46
(11.0%)
9 7 13
(1.3%) (2.5%)
8
(2.1%) (143%)
8 (1.5%) 9 (1.8%) 10 (2.0%) 8 (14%)
A. tONPG.resistant (tONPGr) strains showing full ZacZ activity. j%Galactosidase activity is (see Materials and Methods, section (b)). expressed as units of activity per o.I).~~~ unit of culture B. tONPG-resistant strains showing reduced ZacZ activity. The percentages of the parental levels of p-galactosidase are given.
phenotype expected for true promoter mutants in the fused operon. In order to determine which of the light blue tONPG-resistant colonies were due to mutations in the tyrT gene sequences of the fusion, we tested these strains for recombination with a t*ransducing phage carrying the Su+ allele of the tyrT gene. (b) Recombination
between Lac- fusion m.utants and 48Opsu&
If the mutations causing the Lac- phenotype were in the tyrT gene sequences fused to the lac genes in strain 6, then it should be possible to transduce the mutants to Lac + by recombination with a bacteriophage carrying a wild-type copy of the tyrT gene. The transducing phage +!%Opsu& carries the amber suppressor tRNA allele of the tyrT gene and some surrounding chromosomal material (Fiandt et al., 1971). Integration of this phage via tyrT gene homology-within the tyrT portion of the gene fusion-should yield some Lac + transductants of the mutants (Fig. 1). An important feature of such an integration event is that the mutation responsible for making the fusion Lac- would now be crossed onto the tyrT gene introduced on the transducing phage (see Fig. 1). The arrangement is the result of a single reciprocal recombination event in the promoter proximal sequences of the gene fusion. It was possible to transduce nine independent Lac - mutants to Lac + using @Opst&. These results indicate that in nine mutants, the leaky Lac- phenotype of the fusion is due to mutations in the tyrT gene sequences. These putative promoter mutants were assayed for their effects upon p-galactosidase production in the fusion strains. The results (Table 3B) show that the expression of the fused operon in these mutants is reduced to a few percent of the parental fusion strain. If these transductants arose as predicted, then the suppressor phenotype should be altered in the lysogens (Fig. 1). Since a double crossover event within the tyrT gene would yield Lac+ strains which are not lysogens, only $80 lysogens were tested for suppressor phenotype. In order t,o test whether the suppressor tRNA was expressed at
BOX
II1
I
FIG. 1. Recombination between a fusion strain with a presumptive promoter mutation aml transducing phage 48Opsu&. A transducing phagr> carrying the wild-type Su+ gene is used to infect a strain carrying a tyrT-Zac fusion with a presumptjivc promoter mutation in the tyrT gene sequences. This strain is Zac- and the location of t,hra mut,ation is indicated by X (line 1). A single reciprocal homologous recombination event, in the indicatrtl tyr?’ gene sequences will generate a transductant with reduced suppressor activity. Such a transduct,ant will now become Zac+ (line ~2). Upon ultraviolet irradiat,ion of such a lysogen, $80 prophage excision will occur as follows. I, this excision would occur wit,hin homologous bacterial gene sequences to the right of the tyrT gene. The extent of this material, carried by 48Opsu&,, has been mapped previously (see Fig. 2). 480 phagn from such an excision event will now carry the presumptive promoter mutation. II, t,his excision would occur within tyrl’ genra homology distal to the indicatcxl site of the mutation. The ~$80 phage obtained from such an ox&ion event will now carry a fully active Ru+ gene, and appear identical to the parental +SO~SU&~ phagr. These 2 classes of phage can be distinguished by plaqw color under the appropriate conditions (see Materials and Methods, section (e)). Since t,hc homology available for the second excision event is rather limit,ed, a larger proportion of i3u5 ~$80 phago should be seen. Upon induction of found in t,he lysate. Small open arrows such lysogens, there was from 2 to 4’y0 Su+ phagc represent reduced expression of the indicated genm. I,arg~ open arrows represent wiltl-type or full expression of the indicated genes.
reduced levels, a plate test using a,mber mutants of bact,eriophage T4 was employed (see Materials and Methods, section(d)). A number of T4 amber mutants were screened and three different phage mutant’s were found t,hat showed reduced efficiency of plating on a Lac + transductant of mutant 119, when compared with a related strain carrying the wild-type Su + allele (Table 4). All lysogens of 1#23Opsu&,selected on the basis of 480 immunity, retain full suppressor act,ivity when tested. More than 90% of the Lac+ $80 lysogens tested from each of the nine transduced mutants showed reduced suppressor activity according to the 7’4 phage test. A certain frequency (about 100/b) of the Lac+ #&O lysogenx showed wild-type suppressor activity according to the T4 phage t#est,. These strains may be double lysogens for t)he #~8Opsu,~, transducing phage. or in the process of integration of the phage the mutant allele may have been converted to the wild-type. Gene conversion of this type is frequent when the homology for crossover is extremely limited, forcing the required recombination event, to take place in the immediate vicinity of the mutation (Hotchkiss, 1974).
tRNA
PROMOTER
MUTANTS
TABLE
309
4
Test of suppressor phenotype Strain
T4D
H39
-t
0
T4 phages am17 N133
N319
MBM7007
(Su”)
0
0
MBM7014
(Su+)
t-
f-
/.
+
+
Lac + transduotant
t
$-
0
0
+
(Fig.
0
1)
1 indicates that the phage will grow on the tested strain. 0 indicates no growth. The weak suppressor strains support the growth of the H39 amber mutant, less efficiently than the Su+ wild-type strain. All the T4 mutants used are amber mutants in various phage functions. T4D is t,hr wild-t,ype non-mutant T4 phage.
(c) Further
veri$cation
of the predicted structuw
of the Lac+ tmnsductants
The assumed structure of the Lac+ transducants (Fig. 1) allows predictions about classes of recombinants which might arise from these lysogens. lf the Lac+ 480 lysogens were formed by a single reciprocal recombination event in the tyrT gerle region, then bacterial gene sequences carried by @Opsu& should share homology with adjacent regions on the chromosome. The extent of this homology can be t?stimated from published heteroduplex data (Fiandt, et al., 1971). Recombination within these regions of homology will produce classes of segregants of differing genotypes (Fig. 2). Since the homology for recombination is limit’ed, we used selective techniques based on the predicted Lao phenotype and screened survivors for the other predicted genotypic markers. The majority of segregant-s (classes II, III and TV, Fig. 2) should be resistant to t,ONPG and could be selected on this basis (Table 5). The /3-galactosidase levels of representatives of the different segregants as well as the Lac + t’ransductant of strain 119 are summarized in Table 6. These results are consistent with the structure proposed for the Lac+ 480 transductants.
I!37
I -m -= Pm. 2. (Ilasses (see Fig. 1).
of segregants
Class I, recombination within 1100 base-pairs)?. (‘lass IT, recombination within 2100 base-pairs)& Class III, recombination within (‘lass IV, recombination within large).
from
the Lac + transductants
of the presumptive
homologous
bacterial
gene sequence
homologous
bacterial
gene sequences
promoter
mutants
to the right
of lyrT
(approx.
to the left
of tyrT
(approx.
tyrT gene sequences (extent of homology unknown, but undeleted Mu prophage homology (extent unknown,
limited). possibly
t Estimated from heteroduplox data (Fiandt et al., 1971). $ This prophage material is non-polar on the expression of the Zac genes in the fusion. Genetic analysis of a A transducing phage carrying fusion 6 also indicates the presence of this material IM. Berman, unpublished data).
310
M.
L.
BEltMAN
AND
J.
BECKWITH
5
TABLE
Segregants of a Lac+ transductant Recombinant (Fig.
class
of mutant 119
Genotypc~
2)
h
480
lrrc
Salection
Su
For definition of recombinant classes, see Fig. 2. The genotypes are indicated as follows. For h and $80: + , lysogens; 0, non-lysogens. For Znc : ( , levels same as fusion 6 ; d, Znc genes deleted ; f, weak expression-levels same as mutant 119. For Su: 0, no suppressor activity; j.., reduced suppressor activity. Segregants were selected as resistant to t,ONPG as described. Class III wew selected on tONPG medium in the presence of h clear phagr.
TABLE
/L&lactosidase
levels of various derivatives Suppressor phenotylw
Strain
Vusion
6
Fusion
6, Zac-
119
Su”
508 10
Ru +
Class III segregant (Fig. 2)
Su”
For ,%galactosidase activity, see legend to Table and 3. Su’, non-suppressor phenotype ; Su * , weak
of transducing
of mutant 119 fl-Galactosidaso activity (T/o)
Su”
Lac + transductant (Fig. 1)
(d) Isolation
6
649 10
100 2.1
127 2.1
3. For genotypes of strains, suppressor phenotype.
refer
t,o Figs
1, L’
phage carr?ying the mutant suppressor alleles
Another useful feature of the 9580 lysogens described above is that they permit selection of transducing phages carrying the weak suppressor alleles. The excision events leading to the formation of 480 transducing phage carrying either the wild-type amber suppressor allele or the weak suppressor allele are indicated in the last line of Figure 1 (labelled II and I, respectively). The prophage from the Lac+ 480 transductants was induced by ultraviolet irradiation. As expected these strains yielded phages carrying both full suppressor activity (2 to 4% of total plaque-forming units) or reduced suppressor activity (96 to 98% of total p.f.u.) as judged by plaque color on indicator plates (see Materials and Methods, section (e)). The distribution of suppressor phenotypes agrees with the amount of homology available for the proposed excision events I and II (Fig. 1). The 480 phages carrying the weak suppressor alleles were screened and purified by passage through a h-resistant strain. A X-resistant strain was used since each lysate contained h transducing phage in addition to ~$80 phage. We then tested the ability of lysogens of these 480 phages to suppress a known
tRNA
PROMOTER
3 11
MUTANTS
amber mutation. Strain M7017 carries an amber mutation in the ZacZ gene. !l?hr purified phages were used to make lysogens of M7017 in order to test the level of suppression mediated by the weak suppressor alleles. The level of suppression was determined by measuring the amount of /3-galactosidase produced by the lysogens (Table 7). Lysogens of M7017 were also tested for suppressor activity by using Dhe T4 amber phage test. In the genetic background of strain M7017, the weak suppressor allelt>s could be subdivided into three classes (Table 8).
TABLE 7 levels of lysogens of strain, M7017
p-Galactosidase
p-Galact,osidase
activit)y (% 1
NOdySOgCXl
0.1 I90 50 4 6 5 3 1x 5 5
WPsu,:, &3opsu,;,p74 @opsu,;,pllN #3opsu,:,p27 +3OPsu,:;P45 @opsu,:,p51 @Opsu,?,pB-6 1$8Opsur:lp 1 l-4 +3Opsu,~1p20~:3
(0.05) (100) (26.7) (2.6) (34)
(2.8) (1.6) (9.7) (2%) (29)
Strain JliOli carries the ZncZ amber mutation P14. Lysvgens were obtained as tleacribotl ilr \lateriala and Methods, section (e). The name of t,ho mutant suppressor allele refers to the number tit’ the original tONPG-resistant Lacstrain. The ‘p’ which precetlrs the allele number ~111 hi> usctl to ~+r to promoter mnt,ants.
TABLE
&ualitative
.i llelc
fyrl' (su+) tyrTp74 tyr!Fp119 tyrTp27 tyPTp4B tyrTp51 tyrTp%ti tyrTpll-4 ty?Tp203 Ru”
suppression
test of weak suppressor T4 phagc am17
‘l-411
+
8
i I. poor
v. poor v. poor 1’. poor p00r POW
poor 0
\‘.
x133
alleles
N319
t poor
-t poor
0 0 0 0 0 0 0 0
poor
v. poor v. poor “. poor poor PM” p00r
Class
1 II III III III II II II
0
The same strains described in Table 7 were tested as described in the legend to Table 4. “Poor” and “v. poor” mdicate more suppression than 0, but less than + tyrT indicates the amber srll>pressor allele of the t,RNA gene, and p refers to promoter mutants. 1‘2
3 12
&I.
L.
BERMAN
ANI)
J.
BECKWITH
(e) Backcross of a presumptive promoter mutation from the transfer RNA gene onto the parental fusion In the experiment described below, we demon&rated that it is possible to reintroduce the mutation carried by a weak suppressor transducing phage onto the parental tyrT-Zuc gelle fusion. The $80 phage carrying weak suppressor allele 119 should be able to t’ransduce the Lac + fusion strain t,o Lac ~. A single reciprocal recombination event would accomplish this and render the lysogen f&y Su+ (Fig. 3). Using this 480 phage we conducted a low multiplicity infection of fusion strain 6. Lac- transductants were selected as resistant to tONPG. The Lac- $80 lysogens tested from this cxperiment, 15/18 (83o/o). showed fuZZ suppressor activit,y as determined by the T4 amber phage test,. Thus, restoration of full suppressor activity was associated-for thtx majorit,y of transductants-with lowering the activity of the fusion.
FIG. 3. Recombination between the parental fusion strain ant1 a transducing phage carrying a weak suppressor allele. A transducing phage carrying a weak suppressor allele is used to infect thP parental Znc+ fusion strain (line 1). A single reciprocal recombination event in the indicated tyr7’ gene sequences will generate a transductant with wild-type suppressor activity which has become Zrtc- (line 2). These transductants were selected as t,ONPG-resistant colonies (see text). Small open arrows represent reduced expression of the indicated genes. Large open arrows represent miltl-type or full expression of the indicated genrs.
4. Discussion In this paper we report. the isolation of muta,nts in the t?yrT gene which have all the In a subsequent paper, direct evidence (DNA properties of promoter mutations. in the tyrT sequence analysis) will be presented that these are indeed mutations gene promoter. These mutations were detected by their property of lowering the expression of the Zac genes in a tyrT-lac fusion st’rain. These mutants have properties similar to the promoter mutants isolated in the wildtype Zac operon (Hopkins, 1974). That is, with the exception of one mutant, the expression of the tyrT gene and the tyrT-Zac fusion are lowered to a similar extent (30 to loo-fold). Although the promoter mutations in the wild-type Zac operon fall within two distinct regions of the promoter, all but one of the mutants produce 3 to 6% of the wild-type levels of enzyme. In the case of mutants from the wild-type
t>RSrZ
PROMOTER
MUTANTS
31.7
lac operon, as well as the tyrT-lac fusion, changes which completely abolish promoter activity would not have been selected since the procedure used requires residual expression of the 1acZ gene for screening purposes. The fact that the majority of mutants isolated in t!he present study reduce the level of expression of the fusion to a similar extent ma-y indicate the limits of the screening procedure or that “hot spots” for mutations may exist in the tyrT gene promoter. Based upon careful analysis of the effect, of the mutations on the expression of the suppressor allek of tyrT, the mut’ank can clearly be divided into three distinct classes. Class 1 (represented by 1 mutant) affects the expression of the tRNA gene only moderately, while classes II (4 mutants) and III (3 mutants) are progressively more def(&ve (Table 8). These classes may represent’ different, sites within the tyrT ~~‘rnepromoter. P The genetic technique discussed in Result’s, section (b), can be applied to any fused operon to dctlermine if mutations lie within the gene thought to control the expression of t’he fusion. Th(h basis of this approach is to show that selected alterations in the activit’y of a fusion can be rest’ored to the original activity by recombination with a wild-type copy of one of the genes involved in the fusion. For example, if gene A is fused to and controlling the expression of gene B then mutants selected on the basis of alteratJions in gene B activity can he either in gene B sequences or in gene A sequences. It should be possible to distinguish between these two possibilities by testing whcthcr the mutations recombine with wild-type alleles of either gene. In particular. it should be possible to restore the original B activity of the fusion by recombination with a wild-type copy of gene A if, and only if, the mutations lie \\,it,hin gt~ti(~ ;1 scxcluences fused to gem. B (Fig. 4). If such a cross is performed as
Phoge or episome genes
Frc:. 4. A gvnc?tic cross which localizes mutations within a gene fusion. In the example illustrated, a transducing phage or episomc carrying a wild-type copy of gene A4 is introduced into a strain carrying a fusion of gone A to gene B (@a-B)?. In the example illustrated the fusion does not> C~X~IY~SSgone activity duo to a mutation in the promoter of gene B (X). This type of analysis can also be applied to other classes of mutat,ion in gene 9 which alter the expression of the fusion. ‘l’he recombinant, IS selected based upon restoration of gene B activity. Note that the mutation originally located within the gene A sequences of the fused opemn, is now recombined onto the wild-type all& of gene A and affects the activity of this gene. t It is also possible to perform this cross hdwern a phage or episome carrying the fusion and A wild-type gene on the chromosome.
3I4
31. 1~. HERMAN
d?;L)
.I. li’ECI<~V:I’l’H
depicted in Figure 4, t’he reciprocal produ& are recovered, \vhich results in the mutation being recombined onto the wild-type copy of the gene A. This is a considerable advant’age and allows one to t,est the effect, of mut,ants selected in gene fusions upon t’he expression of the original target opwon (see Results, s&km (b)). LOW suppressor activity is a property expected from mutations wit,hin the (tyry’) gtsnc promoter. However, point mut,ations within the structural gene sequences of this t,RNA also reduce suppressor activity (Smith, 1976). In some cases t,he activit) of the t#RNA molecules produced by the mutant’s is reduced. \vhile in other mutants fully active suppressor tRNAs are produced in much louver amounts due to c!ffects on clearly lie ll;ithin the t,RSA genca ItiVA processing. In all cases these mutal’ions structural sequences. Further: mutation in the fyr?’ structural gent are unlikely t,o afftd the expression of the Iwc genes in a t!/rT-lnc fusion. Recent,ly, DNA sc’quencr informat’ion from t,he region immediat)ely prrccdin g the initiation of t’ranscription of the tyrT gene for the mutants described in t,his paper has been obtained (M. Bermall Rr A. Landy: unpublished results). The results of bhis analysis sho\v that a number of tllcl mut,ants are single base changes within thcl promoter region of the f!/rT gene. A particular benefit, of using gene fusions for genetic ana,lysis is t,lie potential to c>sttnd the range of regulatory effects which ran be tletected. In the case of t,he mutants of t’he suppressor gene described hcbrci. certain alleles clearly retain substantial suppressor activity. For example, the suppressor activity expressed by mutant allele t!/r-l’p74 in many t’ests of suppression cannot be distinguished from the a&ivity of t,hr \iild-t’ypc su + allele. There would have been fe\\ ! if an)-> \\ a>-s to detect such a weak cff%ot~ on t,l:c expression of this gene bv simply tnutagenizinp the Sn + strain and screening 8u - mutant,s. ‘1’11~vctrification of fusions t’o gents n-it,h poorly untkrstootl regulat’ions is difficult, I I) tlrca abscxncc of such knowledge. genetic vctrification of the stru&ure of the fiised opwn must bo obtained. In t’hc preceding paper WV showed that certain mutat’ions \\-hich affect the expression of the wild-type allele of the tyrT gent will a,lso affect, caxpression of luc genes fused to this allele. In t>ltis paper \\y’ show the converse, that wtl could select mutations which affect Uir t~sprc~ssion of the b0c grnc~ in a fusion strain and demonstrate that’ some of t’hesr mutat’ions will affect the expression of the tR1\;A gcnc. In any system, where it is possible to isolate these two classes of mutants, this constitut,cs strong genetic evidence that, t \VO genes are fused int)o a single operon. Since t>he fusions as const’ruct’ed have a complete ba&eriophage genome at a ritr adjacent, on the chromosome, it, is possible to isolate tmnsducing phages carrying thcb Lac A fusions (see preceding paper). l’hrsc phages can bc used to extend the genetic the fusctl niid physiological analysis of the fusions. For cxamplt~? by introducing op~ron into mutant, cells or cells growing in various conditions it is possible to monitor the product,ion of /3-galactosidase by a const)itutively controlled gene fusion. At> present v ~1arc using a technique similar to that described previously (Bchedl & Primalrot?‘. 19X3) to introduce fusion 6, vice phage infect,ion, into colonies gro\l’ti on lactose iudicator plates. (In such a way, \ve expect that the tyrT-bat fusion described here will bc useful for isolat’ing mutants conditionally defective for tRNA or &able RNA regulation.) The isolation of promoter mutants and revtrtants of these mutants has allowed a tlrtailcd model t#o be proposed for the cont,rol of t,he Zac operon (Beckwit,h, 1970; .Dickson ut nl., 1975). This is the first, report of promoter mutants in a gene which code:: for a stable RNS t,ranscript. Mutational analysis of the promot*er site of the
tKN.4
1’ROBIOTKJH
nIr:‘l’i\NTS
::1 .-I
fyrT gem: (as defined by the types of mutants
isolated in the present study) should provide informat,ion about sequences in the DNA critical t,o RNA polymerase interactions. The results of the present stud,y will serve as the basis for coutinued genetic analysis of tRNA biosynthesis in E. coli. ‘I’lris rrsoarcl~ was snpport,ed by a National (:rallt and N.I.H. research grant no. GM13017 for cvwcfully wstiitlg the ma,nnxcript
Institutes of Health Pre-doctoral Training to onr of 11s (.J.B). We tllank Dr Kiels Fiil
REPEKENCES t~acl~rnarur. 13. qJ.. Low, K. B. & Taylor, A. L. (1976). Ha&rid. Rev. 40, I I6 -167. ticckuitl~. .I. K. (1!)70). In Th,e Lac Operon (Beckwith, J. & Zipser. D., eds). pp. 5 -26. (.!oltl Spring Harbor Laboratory, Cold Spring Harbor, New York. f+rman. M. I,. & Reckwith, J. (1979). J. MoE. Riol. 130, 285-301. Hismxs, I’. K. $ Gorini, L. (1972). J. Mol. Biol. 64, 119-134. Hranner. S. & Beckwith, J. (1965). J. Mo2. Biol. 13, 629~--637. (lampbcll. A., Campillo-Campbell, A. & Barker, D. (1978). .I. Racteriol. 135, 9Ok!18. (Iashel, M. (1975). .Iwwu. Rev. Microbial. 29, 301-318. JXharbonill& M., Shaman, H. A., Rilhavy, T. .J. & Schwartz. M. (1978). ./. Xol. BioT. 24. 3.59 3; 1 J)ickson, R. (I., Abelson, J ., Barnes. W. M. & Heznikoff, V+:. 8. (1975). Science, 187, 27-35. l)ickson, 1%.(I.. Abelson, J., .Johnsorr, I’.. Iteznikoff, W. R. & Barnes. W. M. (1977). .J. .&lo’. Hit//. 111 3,65 87. I I”iandt, M.. Hradccna, Z.. Lozeron, H. A. & Szybalski, \Ir. ( 1971). in The Hacteriopha{gcJ Lambda (Hershey, A. D.. ed.), pp. 329-354, Cold Spring Harbor Laboratory. Cold Sprilrg Harbor, New York. Hopkins, .I. 7). (I 974). J. AWol. Biol. 87. 715-724. Hotcllkiss. Ii. D. (1974). zIn.n;u. Rev. Microbial. 28, 445-468. Miller. .I. (1!172). Eqeriments in, .Wolecular Genetics. Cold Spring Harbor Laboratory. C!oltl Spring Harbor, New York. Kezr~ikoff. \V. S. & Thornton: I(. P. (1976). J. Bacterial. 109, 52ti-53%. It~xsrll. R. I,., Abc~lson, J. M., Landy, .-\.. Grfter, M. L., Brenrwr, 8. & Smith, J. D. (1970). .I. .Ilol. Riol. 47, 1~-13. Sclledl, I’. & Primakoff, P. (1973). Proc. Sat. dead. ,Sci.. U.S.A. 70. 2091-2095. Brnitll, .J. D. (1976). In Prog. Xucl. Acid Rea. Mol. Rid. ((‘ok~rr. \V. E., ~(1.). vol. Iti, pp. 2.5 73. .-\cadrlnic: Press. New York.
(1979) 130, 303-315
Use of Gene Fusions to Isolate Promoter Mutants in the Transfer RNA Gene tyrT of Escherichia coli MICHAELLBERMANJ~AND
ijepartment
.JON BECKWITII
of Microhioloyy and Molecular Harvard Me&d School Boston, Mass. 02115, U.S.A.
Genetics
(Received 30 October 1978) LIL tllcl prwed~ng paper. we tlmcribt~ tjtlc isolatiorl of stmins ill whicll tile lac genes are fItsed to and under the control of the structural gene for tyrosine tRNA, t!/r!Z’. Using a wioction for mutants wllictl have lowered expression of the lac genes in sue11 fusion strains, we ha\re detrctrd derivatives whictl carry mutations in ttw t?yrT promoter. These mutations result in a reduction in expression of tile lac genes irl the tyrT-Zac fusion strain to a few prrcent, of tlrr parent levels. These mutations vc~ldtl tw genetically recombirlcd orlto a copy of ttle tyrT germ \v-llich carries ali amber suppwssor allele. Amber sl>ppressor dorivat,ivns wtiictl carry these promotw mutations are greatly reduced in tlloir ability to suppress amber tntltat,ions. consist,ent, with a reduction in transcript,ion of the tyrT gent. Based upon thei int,o three effects on oxpression of the tyrl’ getw, the mutants could be divided clnsscs. Thrw are tho first promoter Instants irl a st,abla RNA gene.
1. Introduction In the prectding paper (Berman & Beckwith, 1979) we showed that it is possible to fuse genetically the transcription controlling sites of a transfer RNA gene, tyrTS> to the DXA sequences which code for the enzymes of the lactose operon (lac). The expression of the structural gene for ,%galactosidase (la&) by the fused hybrid operon is cont,rolled co-ordinately with stable RNB synthesis in Escherichia co&. Insertion tnutat,ions which were isolated as inactivating the tyrT gene were shown to inactivate ctxpression of tjhe Zac genes from the fused operons. In a similar genetic approach WC now show that mutations which are isolated as inactivating expression of a fused operon will, following the appropriate genetic crosses, affect expression of the tyrT Ktme. .4lt,hough numerous mutations within the tyrT gene have been isolated, no mutations affecting tht regulation of this stable RNA gene are known (Smith, 1976). I’sing a fused tyrT-lxx operon for genetic studies, we have SCated studies to select t To whom correspondence should be sent at : Section of Microbiology I )Ivision of Biology and Medicine, Brown TJniversity, Providence, R.I.
$ The genetic
abbreviation3
used arc according
to Rachmann
and Molecular 02912, U.S.A.
et al. (1976). lyrT
Biology.
is t,he gcwt*
coding for the minor species of tyrosine tRNA. The amber suppressor allele of this gene was used throughout,. This allele has been called Ru,,,, su3 as well as sup?’ previously. A bact,eriophagc: carrying this gene (@Opsu&) has bern isolat~c~d previously (Russell et rrl., 1970). Su’ indicates suppressor phenotype, Su” non-suppressor phenotype, and Su * weak suppressor phenotypta. XG, .5-t~romo-4-chl~~~~~-~~-i~~~~~lyl-~-n-gala~~~~si~~~: tONPG, 0.nitrophenyl-p-D-t,hiogalactoside. 303 0(,1’2--“8313/79/150303-
13 $02.00/O
0
1978 Acaclcrnic
I’ress Inc. (London)
Ltd.
304
31. I,. .BERMAN
ANI)
.J. HECKWITH
mutations within the tyrT gene and possibly in genes for other factors which control the t.ranscription of the tRNA gene. Gene fusions to the tat operon have been used previously to obtain mutants affecting the expression or the regulation of hybrid fusion operons (DBbarbouillB et al., 1978; Campbell et al., 1978; Reznikoff & Thorntou. 1972). Here we report the isolation of promoter defective mutants of the tyrT gent’.
2. Materials and Methods strains (a) Hacterial l?usion strain 6 was constructed as described irt the preceding paper. This strain carries a fusion of the lac.2 and ZacY structural penes t,o the controlling region of the tyrT gene. The mutants described in the present study were all isolated from this fusion strain. The parent of this fusion strain was derived from strain M7017 from the collection of J. Beckwith. The genotype of strain M7017 is: F-~ucZ,,,~,,. trpnm3 (A’). SIP. relA. Ot,her bacterial strains have betn described in the preceding paper.
The genetic techniques and t,ht, assay of /3-palactosidase have been described ceding paper and by Miller (1972). The cl>-c, 5-l~ro~no-4-c~~loro-3-indolyl-~-~-~alactoside a substrate for /3-palactosidase, the product of t,he lacZ gene. The enzyme cleaves less compound to produce the blue dye indigo. TIIP production of 8.galactosidase strains correlates well with the degree of blne ~olr)r of colonies on minimal plates 40 pg of this dye/ml. (c) o-Nitroph.erc.yl-8-D-t//ioyalactoside
in the prcis this colorby various containing
selectiorc
Spontaneous mutants resistant, t)o tONP(: \VCLI‘Vselectjet 011 succirlate (0.20,,) minimal plates containing lo-” $1.tONPG and 2Opg X(:/~ul. ‘I’lle pritlciple of tllis selection is described in Kesrllts, section (a). Tllr selection was couductjed a.t 30°C for 3 to 5 days. The level of expression of t.hr lac genes in fusion strain 6 is hiyll enough to inhibit the growth of this strain on this selective medium. Survivors appeared at a frequency of 2 to 3 x 1V7. The selection is based on t,hr method of Hopkius (1974). (d)
1’4
amber
p//aye
test
the ability of strains to support t,lrct Suppressor plienot’ypc* can be tested by ohsen-ing ggowth of T4 phage amber mutants. A fresh Hershey agar plate is overlaid wit,h a lawn of the strain t.o be test,edusing Hershey top agar (Biswas nt Gorini, 1972). Appropriate dilutions of the phage lysates are applied in patclles wit11 tile aid of paper st,rips as described pre1965). In all test,s a wild-type T4 phage is included as well viously (Brenner &z Beckwith, T4 mutants wer<> as control plates seeded with an Su” and an Su+ strain. Pour different obtained from the collections of L. Gorini and H. Wanner and proved to be the most useful for this analysis (Table 1). These phages were used at. a concentration of about lo5 plaqu+ forming units/ml. Weak suppressor strains support varying amounts of lysis when comT4D phage. pared with Su + strains. Su” strains will only allow growth of the wild-type
(H) l,&atior/.
of tra.nsduciny
phage carr,ying
the weuk mppressor
allelex
described in this study were Specialized transducing phages currying t’ht? Inutations isolated as recombinants with the plaque-forming ptlage @OpsuG, (Russell et al., 1970). This phage which carries the amber suppressor allele of tyrT was kindly provided by .J. Smith. As described in Results, section (d) (see Fig. 1) transducing phages carrying the presumptive> promoter mutations are obt.ained in Iysates cont,aining the parental phage as well. The by allowing plaques to suppressor activity of transducing phages can he monitored form on la,ctose indicator plates seeded witlk a strain carrying a lactose amber mutation. 1972) allows a distinction t.o be made bcThe use of tet,razoliurn indicator plates (Miller. tween a phage carrying a weak suppressor allele and a phage carrying t,he wild-type sup) is uscld as an indicator strain on fresh plates, pressor allele. When strain M7017(l~cZ,,~~*
tRNA
PROMOTER TABLE
305
MUTANTS
1
T4 phage ckmIlent
Phagr
-
~,,r dgbtails SW Mat,&&
T4L)
Wild type,
T4 amli
(‘atalytic
requirctment
T4‘amH39
(‘atalyt,ic
requirement
T4 amNl33
Stoichiometric
requirement
T4 amN3 19
Stoichiometric
requirement
and Met~hotls, sert,ion ((I).
ptiage produce red plaques, while phage carrying the Su” allele producr St1+ t,ransdlxing \\hite plaques, and Su * (weak) transducing phage produce light red plaques. The confluent llac- laun is whit<>, which is the reverse of the usual color reaction on this medium. Ptlages carrying weak suppressor alleles were used to lysogenize strain M7017 in t,trc, following way. Since @Opsu,,,+ has a defective attachment site, this bacteriophage lysogerlizes host cells ver>. poorly. In order to select stable lysogens, LO3 cells were infected at R Innltiplicit,y of infection of 0.1 and drown to saturation in I, broth (Miller. 1972) ox-ernightj. Lysope~ls were isolated from the difl’erent cultures by st,reaking the cells 011 lactose MarConkey indicator plates in the presence of @Oc22, a clear mutant of 480. Only cells irmnrrfw to 480, i.0. lysopens, will form colonies on these plates. Lysogens of the phages indicator plates, yet appear carrying the weak suppressor alleles are Lac + on MacConkey Lac - on the less sensitive tetrazolium indicator medium. These lysogens also grow poorl? on minimal plates in the absence of added tryptophan (M7017 is trpam). In contrast. lyso~enq of #W~SII,+,, are fully Lac’ and Trp + on all media tarsted.
3. Results (a) Selection of Lac - ,mutants of &ion.
strain 6
In the preceding paper we described the construction and properties of strains carrying fusions between the tyrT gene and the structural genes of the lac operon. lTsing selective techniques based upon the expression of the lac enzymes, it should be possible t,o isolate from a fusion strain Lac- mutants which are due to defects in the regulation of the tyrT gene. Such mutations could be within the site controlling transcription initiation of the fusion (i.e. within the tyrT gene regulatory sequences) or mutations in other genes required for tyrT expression. Among mut’ations linked to the tyrT-lac fusion, insertions, deletions, and other chromosomal rearrangements disrupting transcription from the tyrT promoter could be selected. It should be possible to recombine such mutations onto a wild-type copy of the tyrT gene and reduce its activity. Small deletions or point mutations within the RNA polymerase binding site would be ext’remely useful in defining the funct,ions of this region of the tyrT gene. We chose a procedure which has been used previously to select, mutants of the wild-type lac operon (Hopkins, 1974). hmong these mutants of the Zac operon, a number have been shown by DNA sequence analysis to be point mutations in the promoter site (Dickson et al., 1975,1977). One of the tyrT-Zac fusion strains described previously was used to select similar mutants of the tyrT gene regulatory region.
3Ot-i
M.
L.
BERMAN
ANI)
.I.
13ECRWT’I’H
The selection t’echnique used is based on the fact that, Lac + strains arr senxitivt~ to the t’oxic lactose analogue O-nitrophenyl-p-n-thiogalactosida (Hopkins, 1974). This compound is transported by the lactose permease. the product of t#he second gene of the Zac operon (lacy). Since the ZacY gene product is required for transport, of t,ONPG int)o the cell, strains wit,h lowered levels of the permease will bc resistant to this cornpound. Among such tnutant’s are certain pleiot.ropir classes which have reduced levels of /?-galact’osidase as well as lact,ose permeasr. Among such mutants in t,he \\ild-t?;pcs Znc operon were mutant)s in the promoter site (Hopkins, 1974). Using this selective technique, we obtained survivors and screened t,hese co1onic.s for reduced expression of t,hc 1acZ gene in th(A following way. Fusion strain 6. a tyrT-Zac + fusion strain. produces up to 500/, of t,hc levels of Zac enzymes compared to the wild-type Zuc operon, and is sensitive to tONPG. From this strain. sponbaneous mut’ant’s resistant t,o tONPG were selected on plates containing the indicator dye X’G (see Materials and Methods. se&ion (c)). This indicator dye yields a blue color when cleaved by /?-galactosidase, making it. possible to screen survivors for enzyme production based upon t,he color of individual colonies. Spontaneous survivors appeared at8 a frequency of 2 to 3 >( lW1. Mutant strains which gave rise to light’ blue colonies. indicating leaky expression of the ZacZ gene, were purified from a number of independent, cultures of isogenic ,wZ.~+ ant1 YPZJ- clerivat(ives of fusion strain li (Table 2)t. !~ABLE 2
tONPG-rehstant
Spontaneous survivors tluction direcbly gene (see test).
from on the selective
mutan.t.s qf tyrT-lac
20 indrpendent, metlium. “Light
sfusion G
vultuwa wtm~ scrrenctt for /3-galactosidase pry,rcducetl cqwession of the ZacZ blw ” indicates
As expected, the majority of the survivors of exposure to tONPG were either Zac 1’ mutants, Lac+ tONPG resistant. or ZacZ mutants polar upon the expression of the distal ZacY gene. The first two classes of mutants form dark blue colonies on the selective plates in the presence of XG. These strains can be shown to produce the same levels of fi-galactosidase as the parent fusion strain (Table 3A). Generally, the third class of mutants form white colonies on XG plates. A number of these mutants proved to be amber, ochre, or UGA mutations in la& (data not shown). However, certain polar mutants in la& might retain adequate levels of fl-galactosidase production to form light blue colonies on the selective medium used. These mutants have the same t Mutations in the rel allele are known to affect stablo RNA gene control under a number ot culture conditions (Ceshel, 1975). Since this selection involves growth on selective plates containWOPC used to avoid any contribution of ing a poor carbon source, both reZA+ and reZ.4 - &ttins the state of the reZAgene to t,he selwtion.
tRNS
PROMOTER
so7
MUTANTS
TABLE 3 p-Galactosidase
levels of tONPG-resistant
mutants
/I-Galactosidase
Strain A. Fusion Fusion Fusion
6, ml+ /relA 6, lmY 6, lot + , tONPG’
B. Fusion Fusion Fusion Fusion Fusion Fusion Fusion Fusion Fusion
6, 6, 6, 6, 6, 6, 6, 6, 6,
ml+, ml+, T&I,
Zue-74 Zuc-119 Zac -20
relA, TelA, relA, relA, relA , relA,
h-27 lnc-45 lnc-51
h-9-6 Zac- 1 l-4 h-20-3
activity
536 480 551
46
(11.0%)
9 7 13
(1.3%) (2.5%)
8
(2.1%) (143%)
8 (1.5%) 9 (1.8%) 10 (2.0%) 8 (14%)
A. tONPG.resistant (tONPGr) strains showing full ZacZ activity. j%Galactosidase activity is (see Materials and Methods, section (b)). expressed as units of activity per o.I).~~~ unit of culture B. tONPG-resistant strains showing reduced ZacZ activity. The percentages of the parental levels of p-galactosidase are given.
phenotype expected for true promoter mutants in the fused operon. In order to determine which of the light blue tONPG-resistant colonies were due to mutations in the tyrT gene sequences of the fusion, we tested these strains for recombination with a t*ransducing phage carrying the Su+ allele of the tyrT gene. (b) Recombination
between Lac- fusion m.utants and 48Opsu&
If the mutations causing the Lac- phenotype were in the tyrT gene sequences fused to the lac genes in strain 6, then it should be possible to transduce the mutants to Lac + by recombination with a bacteriophage carrying a wild-type copy of the tyrT gene. The transducing phage +!%Opsu& carries the amber suppressor tRNA allele of the tyrT gene and some surrounding chromosomal material (Fiandt et al., 1971). Integration of this phage via tyrT gene homology-within the tyrT portion of the gene fusion-should yield some Lac + transductants of the mutants (Fig. 1). An important feature of such an integration event is that the mutation responsible for making the fusion Lac- would now be crossed onto the tyrT gene introduced on the transducing phage (see Fig. 1). The arrangement is the result of a single reciprocal recombination event in the promoter proximal sequences of the gene fusion. It was possible to transduce nine independent Lac - mutants to Lac + using @Opst&. These results indicate that in nine mutants, the leaky Lac- phenotype of the fusion is due to mutations in the tyrT gene sequences. These putative promoter mutants were assayed for their effects upon p-galactosidase production in the fusion strains. The results (Table 3B) show that the expression of the fused operon in these mutants is reduced to a few percent of the parental fusion strain. If these transductants arose as predicted, then the suppressor phenotype should be altered in the lysogens (Fig. 1). Since a double crossover event within the tyrT gene would yield Lac+ strains which are not lysogens, only $80 lysogens were tested for suppressor phenotype. In order t,o test whether the suppressor tRNA was expressed at
BOX
II1
I
FIG. 1. Recombination between a fusion strain with a presumptive promoter mutation aml transducing phage 48Opsu&. A transducing phagr> carrying the wild-type Su+ gene is used to infect a strain carrying a tyrT-Zac fusion with a presumptjivc promoter mutation in the tyrT gene sequences. This strain is Zac- and the location of t,hra mut,ation is indicated by X (line 1). A single reciprocal homologous recombination event, in the indicatrtl tyr?’ gene sequences will generate a transductant with reduced suppressor activity. Such a transduct,ant will now become Zac+ (line ~2). Upon ultraviolet irradiat,ion of such a lysogen, $80 prophage excision will occur as follows. I, this excision would occur wit,hin homologous bacterial gene sequences to the right of the tyrT gene. The extent of this material, carried by 48Opsu&,, has been mapped previously (see Fig. 2). 480 phagn from such an excision event will now carry the presumptive promoter mutation. II, t,his excision would occur within tyrl’ genra homology distal to the indicatcxl site of the mutation. The ~$80 phage obtained from such an ox&ion event will now carry a fully active Ru+ gene, and appear identical to the parental +SO~SU&~ phagr. These 2 classes of phage can be distinguished by plaqw color under the appropriate conditions (see Materials and Methods, section (e)). Since t,hc homology available for the second excision event is rather limit,ed, a larger proportion of i3u5 ~$80 phago should be seen. Upon induction of found in t,he lysate. Small open arrows such lysogens, there was from 2 to 4’y0 Su+ phagc represent reduced expression of the indicated genm. I,arg~ open arrows represent wiltl-type or full expression of the indicated genes.
reduced levels, a plate test using a,mber mutants of bact,eriophage T4 was employed (see Materials and Methods, section(d)). A number of T4 amber mutants were screened and three different phage mutant’s were found t,hat showed reduced efficiency of plating on a Lac + transductant of mutant 119, when compared with a related strain carrying the wild-type Su + allele (Table 4). All lysogens of 1#23Opsu&,selected on the basis of 480 immunity, retain full suppressor act,ivity when tested. More than 90% of the Lac+ $80 lysogens tested from each of the nine transduced mutants showed reduced suppressor activity according to the 7’4 phage test. A certain frequency (about 100/b) of the Lac+ #&O lysogenx showed wild-type suppressor activity according to the T4 phage t#est,. These strains may be double lysogens for t)he #~8Opsu,~, transducing phage. or in the process of integration of the phage the mutant allele may have been converted to the wild-type. Gene conversion of this type is frequent when the homology for crossover is extremely limited, forcing the required recombination event, to take place in the immediate vicinity of the mutation (Hotchkiss, 1974).
tRNA
PROMOTER
MUTANTS
TABLE
309
4
Test of suppressor phenotype Strain
T4D
H39
-t
0
T4 phages am17 N133
N319
MBM7007
(Su”)
0
0
MBM7014
(Su+)
t-
f-
/.
+
+
Lac + transduotant
t
$-
0
0
+
(Fig.
0
1)
1 indicates that the phage will grow on the tested strain. 0 indicates no growth. The weak suppressor strains support the growth of the H39 amber mutant, less efficiently than the Su+ wild-type strain. All the T4 mutants used are amber mutants in various phage functions. T4D is t,hr wild-t,ype non-mutant T4 phage.
(c) Further
veri$cation
of the predicted structuw
of the Lac+ tmnsductants
The assumed structure of the Lac+ transducants (Fig. 1) allows predictions about classes of recombinants which might arise from these lysogens. lf the Lac+ 480 lysogens were formed by a single reciprocal recombination event in the tyrT gerle region, then bacterial gene sequences carried by @Opsu& should share homology with adjacent regions on the chromosome. The extent of this homology can be t?stimated from published heteroduplex data (Fiandt, et al., 1971). Recombination within these regions of homology will produce classes of segregants of differing genotypes (Fig. 2). Since the homology for recombination is limit’ed, we used selective techniques based on the predicted Lao phenotype and screened survivors for the other predicted genotypic markers. The majority of segregant-s (classes II, III and TV, Fig. 2) should be resistant to t,ONPG and could be selected on this basis (Table 5). The /3-galactosidase levels of representatives of the different segregants as well as the Lac + t’ransductant of strain 119 are summarized in Table 6. These results are consistent with the structure proposed for the Lac+ 480 transductants.
I!37
I -m -= Pm. 2. (Ilasses (see Fig. 1).
of segregants
Class I, recombination within 1100 base-pairs)?. (‘lass IT, recombination within 2100 base-pairs)& Class III, recombination within (‘lass IV, recombination within large).
from
the Lac + transductants
of the presumptive
homologous
bacterial
gene sequence
homologous
bacterial
gene sequences
promoter
mutants
to the right
of lyrT
(approx.
to the left
of tyrT
(approx.
tyrT gene sequences (extent of homology unknown, but undeleted Mu prophage homology (extent unknown,
limited). possibly
t Estimated from heteroduplox data (Fiandt et al., 1971). $ This prophage material is non-polar on the expression of the Zac genes in the fusion. Genetic analysis of a A transducing phage carrying fusion 6 also indicates the presence of this material IM. Berman, unpublished data).
310
M.
L.
BEltMAN
AND
J.
BECKWITH
5
TABLE
Segregants of a Lac+ transductant Recombinant (Fig.
class
of mutant 119
Genotypc~
2)
h
480
lrrc
Salection
Su
For definition of recombinant classes, see Fig. 2. The genotypes are indicated as follows. For h and $80: + , lysogens; 0, non-lysogens. For Znc : ( , levels same as fusion 6 ; d, Znc genes deleted ; f, weak expression-levels same as mutant 119. For Su: 0, no suppressor activity; j.., reduced suppressor activity. Segregants were selected as resistant to t,ONPG as described. Class III wew selected on tONPG medium in the presence of h clear phagr.
TABLE
/L&lactosidase
levels of various derivatives Suppressor phenotylw
Strain
Vusion
6
Fusion
6, Zac-
119
Su”
508 10
Ru +
Class III segregant (Fig. 2)
Su”
For ,%galactosidase activity, see legend to Table and 3. Su’, non-suppressor phenotype ; Su * , weak
of transducing
of mutant 119 fl-Galactosidaso activity (T/o)
Su”
Lac + transductant (Fig. 1)
(d) Isolation
6
649 10
100 2.1
127 2.1
3. For genotypes of strains, suppressor phenotype.
refer
t,o Figs
1, L’
phage carr?ying the mutant suppressor alleles
Another useful feature of the 9580 lysogens described above is that they permit selection of transducing phages carrying the weak suppressor alleles. The excision events leading to the formation of 480 transducing phage carrying either the wild-type amber suppressor allele or the weak suppressor allele are indicated in the last line of Figure 1 (labelled II and I, respectively). The prophage from the Lac+ 480 transductants was induced by ultraviolet irradiation. As expected these strains yielded phages carrying both full suppressor activity (2 to 4% of total plaque-forming units) or reduced suppressor activity (96 to 98% of total p.f.u.) as judged by plaque color on indicator plates (see Materials and Methods, section (e)). The distribution of suppressor phenotypes agrees with the amount of homology available for the proposed excision events I and II (Fig. 1). The 480 phages carrying the weak suppressor alleles were screened and purified by passage through a h-resistant strain. A X-resistant strain was used since each lysate contained h transducing phage in addition to ~$80 phage. We then tested the ability of lysogens of these 480 phages to suppress a known
tRNA
PROMOTER
3 11
MUTANTS
amber mutation. Strain M7017 carries an amber mutation in the ZacZ gene. !l?hr purified phages were used to make lysogens of M7017 in order to test the level of suppression mediated by the weak suppressor alleles. The level of suppression was determined by measuring the amount of /3-galactosidase produced by the lysogens (Table 7). Lysogens of M7017 were also tested for suppressor activity by using Dhe T4 amber phage test. In the genetic background of strain M7017, the weak suppressor allelt>s could be subdivided into three classes (Table 8).
TABLE 7 levels of lysogens of strain, M7017
p-Galactosidase
p-Galact,osidase
activit)y (% 1
NOdySOgCXl
0.1 I90 50 4 6 5 3 1x 5 5
WPsu,:, &3opsu,;,p74 @opsu,;,pllN #3opsu,:,p27 +3OPsu,:;P45 @opsu,:,p51 @Opsu,?,pB-6 1$8Opsur:lp 1 l-4 +3Opsu,~1p20~:3
(0.05) (100) (26.7) (2.6) (34)
(2.8) (1.6) (9.7) (2%) (29)
Strain JliOli carries the ZncZ amber mutation P14. Lysvgens were obtained as tleacribotl ilr \lateriala and Methods, section (e). The name of t,ho mutant suppressor allele refers to the number tit’ the original tONPG-resistant Lacstrain. The ‘p’ which precetlrs the allele number ~111 hi> usctl to ~+r to promoter mnt,ants.
TABLE
&ualitative
.i llelc
fyrl' (su+) tyrTp74 tyr!Fp119 tyrTp27 tyPTp4B tyrTp51 tyrTp%ti tyrTpll-4 ty?Tp203 Ru”
suppression
test of weak suppressor T4 phagc am17
‘l-411
+
8
i I. poor
v. poor v. poor 1’. poor p00r POW
poor 0
\‘.
x133
alleles
N319
t poor
-t poor
0 0 0 0 0 0 0 0
poor
v. poor v. poor “. poor poor PM” p00r
Class
1 II III III III II II II
0
The same strains described in Table 7 were tested as described in the legend to Table 4. “Poor” and “v. poor” mdicate more suppression than 0, but less than + tyrT indicates the amber srll>pressor allele of the t,RNA gene, and p refers to promoter mutants. 1‘2
3 12
&I.
L.
BERMAN
ANI)
J.
BECKWITH
(e) Backcross of a presumptive promoter mutation from the transfer RNA gene onto the parental fusion In the experiment described below, we demon&rated that it is possible to reintroduce the mutation carried by a weak suppressor transducing phage onto the parental tyrT-Zuc gelle fusion. The $80 phage carrying weak suppressor allele 119 should be able to t’ransduce the Lac + fusion strain t,o Lac ~. A single reciprocal recombination event would accomplish this and render the lysogen f&y Su+ (Fig. 3). Using this 480 phage we conducted a low multiplicity infection of fusion strain 6. Lac- transductants were selected as resistant to tONPG. The Lac- $80 lysogens tested from this cxperiment, 15/18 (83o/o). showed fuZZ suppressor activit,y as determined by the T4 amber phage test,. Thus, restoration of full suppressor activity was associated-for thtx majorit,y of transductants-with lowering the activity of the fusion.
FIG. 3. Recombination between the parental fusion strain ant1 a transducing phage carrying a weak suppressor allele. A transducing phage carrying a weak suppressor allele is used to infect thP parental Znc+ fusion strain (line 1). A single reciprocal recombination event in the indicated tyr7’ gene sequences will generate a transductant with wild-type suppressor activity which has become Zrtc- (line 2). These transductants were selected as t,ONPG-resistant colonies (see text). Small open arrows represent reduced expression of the indicated genes. Large open arrows represent miltl-type or full expression of the indicated genrs.
4. Discussion In this paper we report. the isolation of muta,nts in the t?yrT gene which have all the In a subsequent paper, direct evidence (DNA properties of promoter mutations. in the tyrT sequence analysis) will be presented that these are indeed mutations gene promoter. These mutations were detected by their property of lowering the expression of the Zac genes in a tyrT-lac fusion st’rain. These mutants have properties similar to the promoter mutants isolated in the wildtype Zac operon (Hopkins, 1974). That is, with the exception of one mutant, the expression of the tyrT gene and the tyrT-Zac fusion are lowered to a similar extent (30 to loo-fold). Although the promoter mutations in the wild-type Zac operon fall within two distinct regions of the promoter, all but one of the mutants produce 3 to 6% of the wild-type levels of enzyme. In the case of mutants from the wild-type
t>RSrZ
PROMOTER
MUTANTS
31.7
lac operon, as well as the tyrT-lac fusion, changes which completely abolish promoter activity would not have been selected since the procedure used requires residual expression of the 1acZ gene for screening purposes. The fact that the majority of mutants isolated in t!he present study reduce the level of expression of the fusion to a similar extent ma-y indicate the limits of the screening procedure or that “hot spots” for mutations may exist in the tyrT gene promoter. Based upon careful analysis of the effect, of the mutations on the expression of the suppressor allek of tyrT, the mut’ank can clearly be divided into three distinct classes. Class 1 (represented by 1 mutant) affects the expression of the tRNA gene only moderately, while classes II (4 mutants) and III (3 mutants) are progressively more def(&ve (Table 8). These classes may represent’ different, sites within the tyrT ~~‘rnepromoter. P The genetic technique discussed in Result’s, section (b), can be applied to any fused operon to dctlermine if mutations lie within the gene thought to control the expression of t’he fusion. Th(h basis of this approach is to show that selected alterations in the activit’y of a fusion can be rest’ored to the original activity by recombination with a wild-type copy of one of the genes involved in the fusion. For example, if gene A is fused to and controlling the expression of gene B then mutants selected on the basis of alteratJions in gene B activity can he either in gene B sequences or in gene A sequences. It should be possible to distinguish between these two possibilities by testing whcthcr the mutations recombine with wild-type alleles of either gene. In particular. it should be possible to restore the original B activity of the fusion by recombination with a wild-type copy of gene A if, and only if, the mutations lie \\,it,hin gt~ti(~ ;1 scxcluences fused to gem. B (Fig. 4). If such a cross is performed as
Phoge or episome genes
Frc:. 4. A gvnc?tic cross which localizes mutations within a gene fusion. In the example illustrated, a transducing phage or episomc carrying a wild-type copy of gene A4 is introduced into a strain carrying a fusion of gone A to gene B (@a-B)?. In the example illustrated the fusion does not> C~X~IY~SSgone activity duo to a mutation in the promoter of gene B (X). This type of analysis can also be applied to other classes of mutat,ion in gene 9 which alter the expression of the fusion. ‘l’he recombinant, IS selected based upon restoration of gene B activity. Note that the mutation originally located within the gene A sequences of the fused opemn, is now recombined onto the wild-type all& of gene A and affects the activity of this gene. t It is also possible to perform this cross hdwern a phage or episome carrying the fusion and A wild-type gene on the chromosome.
3I4
31. 1~. HERMAN
d?;L)
.I. li’ECI<~V:I’l’H
depicted in Figure 4, t’he reciprocal produ& are recovered, \vhich results in the mutation being recombined onto the wild-type copy of the gene A. This is a considerable advant’age and allows one to t,est the effect, of mut,ants selected in gene fusions upon t’he expression of the original target opwon (see Results, s&km (b)). LOW suppressor activity is a property expected from mutations wit,hin the (tyry’) gtsnc promoter. However, point mut,ations within the structural gene sequences of this t,RNA also reduce suppressor activity (Smith, 1976). In some cases t,he activit) of the t#RNA molecules produced by the mutant’s is reduced. \vhile in other mutants fully active suppressor tRNAs are produced in much louver amounts due to c!ffects on clearly lie ll;ithin the t,RSA genca ItiVA processing. In all cases these mutal’ions structural sequences. Further: mutation in the fyr?’ structural gent are unlikely t,o afftd the expression of the Iwc genes in a t!/rT-lnc fusion. Recent,ly, DNA sc’quencr informat’ion from t,he region immediat)ely prrccdin g the initiation of t’ranscription of the tyrT gene for the mutants described in t,his paper has been obtained (M. Bermall Rr A. Landy: unpublished results). The results of bhis analysis sho\v that a number of tllcl mut,ants are single base changes within thcl promoter region of the f!/rT gene. A particular benefit, of using gene fusions for genetic ana,lysis is t,lie potential to c>sttnd the range of regulatory effects which ran be tletected. In the case of t,he mutants of t’he suppressor gene described hcbrci. certain alleles clearly retain substantial suppressor activity. For example, the suppressor activity expressed by mutant allele t!/r-l’p74 in many t’ests of suppression cannot be distinguished from the a&ivity of t,hr \iild-t’ypc su + allele. There would have been fe\\ ! if an)-> \\ a>-s to detect such a weak cff%ot~ on t,l:c expression of this gene bv simply tnutagenizinp the Sn + strain and screening 8u - mutant,s. ‘1’11~vctrification of fusions t’o gents n-it,h poorly untkrstootl regulat’ions is difficult, I I) tlrca abscxncc of such knowledge. genetic vctrification of the stru&ure of the fiised opwn must bo obtained. In t’hc preceding paper WV showed that certain mutat’ions \\-hich affect the expression of the wild-type allele of the tyrT gent will a,lso affect, caxpression of luc genes fused to this allele. In t>ltis paper \\y’ show the converse, that wtl could select mutations which affect Uir t~sprc~ssion of the b0c grnc~ in a fusion strain and demonstrate that’ some of t’hesr mutat’ions will affect the expression of the tR1\;A gcnc. In any system, where it is possible to isolate these two classes of mutants, this constitut,cs strong genetic evidence that, t \VO genes are fused int)o a single operon. Since t>he fusions as const’ruct’ed have a complete ba&eriophage genome at a ritr adjacent, on the chromosome, it, is possible to isolate tmnsducing phages carrying thcb Lac A fusions (see preceding paper). l’hrsc phages can bc used to extend the genetic the fusctl niid physiological analysis of the fusions. For cxamplt~? by introducing op~ron into mutant, cells or cells growing in various conditions it is possible to monitor the product,ion of /3-galactosidase by a const)itutively controlled gene fusion. At> present v ~1arc using a technique similar to that described previously (Bchedl & Primalrot?‘. 19X3) to introduce fusion 6, vice phage infect,ion, into colonies gro\l’ti on lactose iudicator plates. (In such a way, \ve expect that the tyrT-bat fusion described here will bc useful for isolat’ing mutants conditionally defective for tRNA or &able RNA regulation.) The isolation of promoter mutants and revtrtants of these mutants has allowed a tlrtailcd model t#o be proposed for the cont,rol of t,he Zac operon (Beckwit,h, 1970; .Dickson ut nl., 1975). This is the first, report of promoter mutants in a gene which code:: for a stable RNS t,ranscript. Mutational analysis of the promot*er site of the
tKN.4
1’ROBIOTKJH
nIr:‘l’i\NTS
::1 .-I
fyrT gem: (as defined by the types of mutants
isolated in the present study) should provide informat,ion about sequences in the DNA critical t,o RNA polymerase interactions. The results of the present stud,y will serve as the basis for coutinued genetic analysis of tRNA biosynthesis in E. coli. ‘I’lris rrsoarcl~ was snpport,ed by a National (:rallt and N.I.H. research grant no. GM13017 for cvwcfully wstiitlg the ma,nnxcript
Institutes of Health Pre-doctoral Training to onr of 11s (.J.B). We tllank Dr Kiels Fiil
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