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Locations of ribosome-binding sites in the nin5 region of bacteriophage λ
Gene, 10 (1980) 167-175 © Elsevier/North-HollandBiomedicalPress
167
L o c a t i o n s o f r i b o s o m e - b i n d i n g sites in the n i n 5 region o f b a c t e r i o p h a g e X (E. coli; deletion; single-stranded DNA; mRNA; electron microscopy; transposons; restriction enzymes)
Tom Chiang and Garrett lhler Department of Medical Biochemistry, Texas A & M College of Medicine, College Station, TX 77843 (U.S.A.)
(Received November 16th, 1979) (Revision received and accepted February 26th, 1980)
SUMMARY Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region. These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene R z and another inserted near gene Q. These ribosome binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible X genes.
INTRODUCTION Calame and Ihler (1977a) developed an electron microscopic procedure for locating ribosome binding sites on DNA. Ribosomes are allowed to bind to single-stranded DNA serving as an analog of mRNA and the DNA-ribosome complexes are visualized using the electron microscope. The positions of the bound ribosomes are determined relative to a reference point such as a transposon insertion site or an end created by a restriction enzyme. Most of the ribosome binding sites detected by this procedure may correspond to polypeptide initiation sites used in vivo since binding on X DNA (Calame and Ihler, 1977a,b) or T7 DNA (Ihler and Nakada, 1970) occurs predominantly to that strand of DNA which contains the same base sequence as mRNA. This procedure may be especially useful for Abbreviations: bp, base pairs; %h unit, 480 bp;p, promoter; s, startpoint; t, terminator.
studying regions of DNA for which there is no genetic detection system available. For example, there are several extensive regions in the bacteriophage X genome which are so far devoid of known genes despite the fact that X has been extensively studied by most of the techniques of genetics and molecular biology. One of these regions is the b2 region, defined by the b2 deletion, which is known to code for polypeptides of unknown function (Hendrix, 1971). Another is the region defined by the nin5 deletion and a third is the region between gene R z and the right end of X DNA. We have been locating ribosome-binding sites on bacteriophage X DNA and have now located the ribosome-binding sites on the right-hand 40% of the DNA (Calame and Ihler, 1977b; Chiang et all, 1979). Comparison of the locations of these ribosome-binding sites with the known genetic and physical map of and with the limited amount of DNA sequence data presently available for X DNA, shows a general correlation between the locations of some ribosome binding
168 sites and the locations of polypeptide initiation sites for known genes. Other ribosome-binding sites have been found in genetically silent regions and these may indicate the existence of as yet unknown genes, although definitive proof that these ribosome-binding sites are functional in vivo must await detection of the polypeptides. We have now located nearly forty ribosome-binding sites on the right-hand 40% of DNA, of which sixteen may correspond to known genes.
(c) Preparation of phage T4 gene 32 protein Bacteriophage T4 gene 32 protein was purified from a T4-infected E. coli cell lysate on a denatured calf thymus DNA-cellulose column as described (Alberts and Herrick, 1971; Chiang et al., 1979). Gene 32 protein prevents random base interactions of DNA which would otherwise be fixed by glutaraldehyde and also greatly improves the morphology of single-stranded DNA under electron microscope. (d) Binding of ribosomes to single-stranded DNA
MATERIALS AND METHODS
(a) Bacteria and phage ~in5dk6 phage (~519cI857nin5Sam7Rz: :Tn903) contains the nin5 deletion and a transposable DNA element inserted into gene Rz (Young et al., 1979). Xpk3 (~b519ci857Tn903 Sam7) has the transposon inserted about 3900 bp from the right end. ~pk35nin5 (~b519b515ci857cli: :Tn903nin5Sam7) contains the transposon inserted in gene clI. Phage lysogens, obtained from R. Young (Young et al., 1979), were grown in broth at 30°C to between 5 × 10 a cells/ml to 109 cells/ml before they were thermoinduced for 15 min at 43°C. After an additional 2 - 3 h incubation at 37°C, cells were concentrated by centrifugation and lysed by rapid freezing and thawing. Phage particles were purified by standard CsC1 density gradient centrifugation. ¢X174 single-stranded DNA was obtained by phenol extraction of purified ~bX174 am3 phage by the method of Pagano and Hutchinson (1971). E. coli ribosomes, initiation factors and charging enzymes were prepared from strain 238 (DNase I-, B-) as described by Ihler et al. (1979).
Ribosome-binding reactions were carried out and the DNA-ribosome complexes were purified as described previously (Calame and Ihler, 1977a).
(e) Electron microscopy of DNA-ribosome complexes and of DNA heteroduplexes The DNA-ribosome complexes were fixed, spread and picked up on carbon-coated grids as described by Ihler et al. (1979). DNA molecules with a transposon (containing a stem and loop) and one or more bound ribosomes were photographed. Distances were determined by tracing the projected negative using a Numonics Graphics Calculator (Model 1224, Numonics Corporation). One or more ¢X174 single-stranded DNA molecules in the same field were also measured and used as a length standard. DNA heteroduplexes were prepared and measured by the formamide technique of Westmoreland et al. (1969) as described previously (Chiang et al., 1979).
RESULTS
(b) Preparation of phage DNA for ribosome binding Single-stranded ~,pk3 DNA was prepared from purified phage particles by simultaneous lysis and denaturation in 0.2 N NaOH. The denatured DNA (about 50/ag/ml) was neutralized and dialysed at 4°C against 10 mM Tris, pH 7.4 and 1 mM magnesium acetate. In order to assure complete annealing of the inverted repeat of the transposon, the singlestranded DNA was incubated for 2 rain at 65°C before use in the binding reaction.
In a previous study we determined the number and locations of the ribosome binding sites on the right end of k DNA (Chiang et al., 1979). Those studies utilized knin5dk6, which carries the nin5 deletion, and so we employed another phage, kpk3, which is nin + to determine the number and locations of ribosome binding sites within the nin5 region. Measurements on kninSdk6 DNA were made relative to a transposon insertion site in gene R z (Young et al., 1979). kpk3 contains the same transposon inserted at a different
169
site. To ensure that the locations of ribosome binding sites determined using one transposon as a reference point would correspond closely with the locations determined using the other transposon, we measured the positions cf both transposon insertion sites simultaneously by heteroduplexing DNA from ~.pk3 with DNA from Xnin5dk6. We found the transposon in Xnin5dk6 to be inserted 2310 + 90 bp from the right end of the X DNA (when measuring the heteroduplex molecule and using double-stranded ¢X174 as the length standard), and 2340-+ 330 bases from the right end, when measuring single-stranded Xnin5dk6 DNA using single-stranded ¢X174 as the length standard. This position is in close agreement with the location determined by Young et al. (1979). The transposon insertion site in Xpk3 DNA is located 1590 -+ 170 bp left of the transposon in ?,nin5dk6, corresponding to 3900 -+ 180 bp from the right end of ?~DNA. Assuming 48000 bp for ~t DNA, this corresponds to map position 91.9 %~, which differs significantly from the position of 92.3 %~ reported by Young et al. (1977). Using heteroduplexes between ?~nin5dk6 and ]tpk3, we located the right terminus of nin5 about 5160 -+ 250 bp from the right end of ?t DNA (Chiang et al., 1979). We have now made heteroduplexes between ?~pk3 and ~pk35nin5. For a length standard we use 3900 bp as the distance from the right end of ~ DNA to the Xpk3 transposon insertion site. The transposon insertion sites in Xpk3 and ?~pk35nin5 are separated by 3470 ± 180bp. The right terminus of nin5 is located 1360 + 90 bp from the transposon insertion site in ;kpk3 which corresponds to 5260 ± 270 bp from the right end of X DNA, in close agreement with
pk35
the previous determination of 5160 bp. The left terminus of nin5 is 2110 bp from the transposon in
?tpk35nin5. The nin5 deletion was originally measured to be a 5.4 %~ deletion. More recently Szybalski et al. (1977) have reported nin5 to be a 5.8% deletion with its right end at 89.5 %h, i.e., 5040 bp from the right terminus. Assuming 48 000 bp for ~, DNA (Szybalski et al., 1977), the length of nin5 would be about 2800 bp. We will take the endpoints of nin5 to be located 5200 and 8000 bp from the right end of ~t DNA. The transposon insertion site in ~pk35nin5 is within gene clI, 180 bp from the clI polypeptide initiation site (D. Moore, personal communication). Taking 2800 bp as the length of nin5, this would place the clI polypeptide initiation sites 10350 bp from the right end of ?~ and the gene O polypeptide initiation site about 10000 bp from the right end of ~, DNA. Fig. 1 shows the separation in bp from these various markers. Fig. 2 shows a histogram of the number of ribosomes bound in each 100 base interval for ~v~in5dk6 and ?tpk3. Table I lists the number of bases separating the ribosome binding sites from the transposon insertion sites on ?~pk3 and ?,nin5dk6 and the locations of the ribosome binding sites in bases from the right end of ?t DNA. It can be seen that the measured locations correspond closely for the ribosome binding sites common to the two ~ DNA molecules. For example, measurements on both ~ i n 5 d k 6 and ~.pk3 DNA demonstrate the presence of a ribosome binding site located 4750 bases from the right end of ~. Another ribosome binding site is located 4410 bases from the
dk6
pk3
?
I
nin5
I
1260
2110
.ia "I-
--2000
"-)"
?
?
3',~
1590
"J"
2310
1360
Fig. 1. Distance measurements. The measurements of the locations of the three transposons referred to in the text arid of the right and left ends of the nin5 deletion are summarized here. Two separate measurements for the location of the right end of nin5 were made. The first measurement, made using heteroduplexes between hpk3 and ~ninSdk6, gave a value of 5160 + 250 bp from the right end of h DNA. Double-stranded OX174 (5386 bp) was used as a length standard to determine the position of the transposon in knin5dk6 relative to the right end. A second measurement for the location of the right end of nin5 gave a value of 5260 + 270 bp from the right end of h. This measurement was made using heteroduplexes between kpk3 and hpk35nin5. The previously determined distance of 3900 bp from the right end for the transposon in hpk3 was used as the length standard.
170 TABLE I Location of ribosome binding sites on hpk3 and hninSdk6. The distance of ribosome binding sites from the transposon in hnin5dk6 or hpk3 is given in bases measured using ~X174 DNA (5386 bases) as a length standard. The distance from the right end is calculated assuming that the transposon insertion site in hnin5dk6 is located 2310 bases from the right end and that the transposon-insertion site in hpk3 is located 3900 bases from the right end. Distances for ribosome binding sites on hnin5dk6 are corrected for the nin5 deletion (2800 bases) where appropriate. For the site which could lie on either or both sites of nin5, it was assumed that it lies on both sides and these are the values given (5150, 8150) in the data for hnin5dk6
hnin5dk6
hpk3 Bases from right end
Bases from transposofi
3700 4410 4750 5060 5620 5950 6200 6550 6750 7060 7500 8190 9100-10000
Number of ribosomes
200 510 850 1160 1720 2050 2300 2650 2850 3160 3600 4290 5200-6100
3 5 5 5 2 5 11 I1 8 5 6 2 13
Polypeptide product
Bases from right end
Bases from transposon
Number of ribosomes
220 1165 1520 1980 2590 3010 3410 3670 4260 4770 5150
2090 1145 790 330 280 700 1100 1360 1950 2460 -2900
3 3 8 6 9 3 8 8 9 7 12
8150 9100-10000
-2900 4000-4900
12 20
9000 35000 13000 17000 23000 16000 15000 9600 22000 19000 13000 21000 12000 9200 11000 7400 12000 16000 26000 -
nin5
!
,k6
,"I, 4 g~ end
|
,
nin5
J
6q
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I I
p.k3
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9
8
7
6
5
4
5
2
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0 end
KI LOBASES Fig. 2. Histogram of bound rbosomes. The locations of bound ribosomes on hpk3 DNA and ~inSdk6 DNA were measured relative to the transposons using ~X174 as a length standard. All ribosomes falling into each 100 base increment were pooled for plotting the histogIams. The two histograms correspond for sites located 3700, 4]40, 4750, and 5060 basesfrom the fight end of h. Ribosomes bound to hpk3 DNA between 0 and 3500 bases from the right end of DNA are omitted due to the small number of ribosomes measured in this region. The measured location for the fight end of nin5 is 5200 bases from the fight end of h DNA. nin5 deletes about 2800 bp from hnin5dk6 (upper histogram). This DNA is present in ;~pk3 (lower histogram) in the region located between 5200 and 8000 bases from the rght end of h DNA.
171 right end using ),pk3 DNA and 4260 bases from the right end using Xnin5dk6 DNA. This ribosome binding site may correspond to the polypeptide initiation site for gene Q. We have not a~tempted to extensively locate ribosome-binding sites on Xpk3 tO the right of the transposon since this region has been studied previously (Chiang et al., 1979) but those ribosomes which we have measured fall near ribosome-binding sites found previously except for one ribosome bound about 730 bases from the right end of X. We have confirmed, using Xpk3, the location of a ribosome binding site previously found with Xnin5dk6 in or very near the region from which 6S RNA is transcribed. Within the 6S RNA sequence itself are only 3 AUG codons, each of which has a preceeding sequence which might function as a Shine and Dalgarno (1974) sequence.
right end of )`. The distances separating these ribosome binding sites range from 200 to 565 bases, corresponding to potential polypeptides ranging in molecular weight from 7500 to 21000 if it is assumed ribosome binding sites all correspond to polypeptide initiation sites for different genes (Table I). Since the right terminus of nin5 has been measured as 5160 -+ 250 bp or 5260 -+ 270 bp, it is likely but not certain that the ribosome binding site at 5060 bases from the right end of ), DNA lies to the right of nin5. However, it is certain that the remaining seven ribosome binding sites located between 5620 and 7500 bases must be removed by the nin5 deletion. Assuming 2800 bp for the length of the deletion and placing the right terminus at 5200 bp from the right end of), DNA, the left end ofnin5 would lie 8000 bp from the right end of ), DNA which is well beyond the ribosome binding site located 7500 bases from the right end of X DNA. To confirm this, we have compared the location of ribosome binding sites on )`pk3 and ),ninSdk6 (Fig. 2). To the left of the transposon on )`pk3 are eleven ribosome binding sites. In addition a cluster of perhaps four ribosome binding sites is found between 9100 and 10000 bases from the right end of X. This cluster must lie well to the left of the nin5 region. A similar cluster of ribosome binding sites therefore should be found on ),nin5dk6. Such a cluster can be found located 6300-7200 bases from the right end of Xnin5dk6 or 9 1 0 0 - 1 0 0 0 0 bases from the right end of ), if 2800 bases are added to compensate for the nin5 deletion. We are attempting to measure the location of the ribosome binding sites more exactly by binding ribosomes to ),pk35nin5 DNA, which has
5'-TGGGUAAAUUUGACUCAACGAUG-3' 5'-UGGUAGUGAGAUG-3' 5'-AGAGGUCUGCAAAAUG-3' The AUG codon in the first sequence is followed one codon later by the polypeptide termination codon UAA. The other two are read in the same phase and no polypeptide termination codons occur up to the end of the 6S RNA sequence. Either of these latter two sequences could correspond to the observed ribosome binding site. The second sequence would code for the longer polypeptide. The third sequence has a more convincing Shine and Dalgarno sequence, but would code for a much shorter polypeptide. Eight ribosome binding sites have been located on ),pk3 DNA between 5060 and 7500 bases from the
[
J
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1
nin~
I
•
1
1
1
t~
1
l
1
1
1 , I
,o
I erd
KILOBASES from RIGHT END
Fig. 3. Location of ribosome binding sites. The stem and loop symbols indicate the sites of insertion of two transposons, one in Xnin5dk6 (Rz, 2310 bases from the right end) and one in hpk3 (3900 bases from the right end). Arrows indicate the locations of ribosome binding sites measured in kilobases from the right end of ~, DNA. Arrows in the upper row indicate the location of ribosome binding sites determined using hnin5dk6 DNA, assuming the right and left ends ofnin5 are located 5200 and 8000 bp respectively from the right end of h DNA. Arrows in the lower row indicate the locations of ribosome binding sites determined using hpk3 DNA. Ribosome binding sites on kpk3 between 6S RNA and the fight end of h were not determined. Solid triangles indicate the endpoints of nin5 (5040 and 7823 bp from the right end) as determined by Szybaiski et al. (1977) (see also Szy-
balski and Szybalski, 1979).
172 a transposon inserted in clI very near the polypeptide initiation site for gene O. Fig. 3 shows the position of ribosome binding sites that must lie outside the nin5 region, either to the right or to the left. The only potential ambiguity lies with deciding whether the ribosome binding site located on kpk3 DNA 5060 bp from the right end is or is not removed by the nin5 deletion. The interpretation which we prefer is that the ribosome binding site at 5060 bases is not removed by nin5. The profile of bound ribosomes on )~nin5dk6 between 5000 and 5500 bases from the right end suggests the presence of two close ribosome binding sites. If so, one of these ribosome binding sites is likely to correspond to the ribosome binding site found on ~pk3 5060 bases from the right end of ~. The other is likely to correspond to the ribosome binding site found on ;~pk3 at 8190 bases from the right end of k. On ~nin5dk6 these two ribosome-binding sites would be brought to within 350 bases of each other by the 2800 base nin5 deletion. If it is assumed that the profile of bound ribosomes located 5300 bases from the right end of ~nin5dk6 DNA represents a single ribosome-binding site, this site could correspond either to the site on kpk3 5060 bases from the right end or to the site 8190 bases from the right end. In the former case the end points of nin5 would have to be about 5400 and 8200 bases from the right end of ~. DNA. In the latter case the end points of nin5 would be about 5000 and 7800 bases from the right end. The ribosome binding site located 4770 bases from the right end of ;~nin5dk6 would align equally well with Xpk3 at this position or at a position 7550 bases from the right end. The latter assignment however seems unlikely since it would require that nin5 remove the ribosome binding site located on Xpk3 at 4750 bases from the right end. This appears to be too far from the measured location of the right terminus of nin5 at 5160 + 250 or 5260 + 270 bp from the right end, and even from the 5040 bp location measured by Szybalski et al. (1977).
DISCUSSION In the absence of the X repressor protein, rightward and leftward mRNA synthesis begins on ~ DNA
at two promoters, PL and PR, which are located on opposite sides of the immunity region containing the gene (cI) that codes for the X repressor. The rightward mRNA transcript terminates at the tR~ and tR2 sites located after genes cro and P respectively. The product of the N gene, contained in the leftward mRNA transcript, acts as an anti-termination factor for RNA synthesis, probably by binding to RNA polymerase, and allows mRNA transcription to proceed through tR1 and tR2 to include genes cII, O, P, the nin5 region and gene Q. Gene cII plays a role in the establishment of lysogeny. Genes O and Pare required for X DNA replication. Gene Q codes for a protein which is required for expression of all late X genes and acts at a site termed P'R (or pQ) located to the right of Q. A small RNA molecule, 6S RNA, is synthesized from the region to the right of Q and it has been proposed that the Q protein may act as an antiterminator for the 6S molecule, allowing rightward RNA synthesis to continue from P'R to genes S, R, and Rz, which are required for lysis of the bacterial host, and then to genes coding for phage structural proteins. A general discussion of the molecular biology of bacteriophage ~ can be found in Lewin (1977). The first three genes transcribed from PR, cro, cII, and O, have been sequenced. The polypeptide initiation site for cro is 19 bp from s R and the cro gene is 201 bp long, There are 118 bp between the cro polypeptide site and the polypeptide initiation site of gene cII. cII is 294 bp long (Schwarz et al., 1978) and is separated by 32 bp from the presumed polypeptide initiation site of gene O. Gene O is 897 bp long (Scherer, 1978). The region between genes P and Q is largely devoid of known genes and no polypeptides have been identified which are coded for by genes in this region. Most or all of the genes in this region must be dispensible for normal ~ development since the nin5 deletion removes about 2800 bp between P and Q without affecting viability. The nin5 deletion was isolated as a ~ variant which could grow in the absence of gene N product (Court and Sato, 1969). Apparently an N-sensitive terminator(s) for mRNA synthesis exist(s) in this region, between P and Q. The existence of this terminator cannot be readily demonstrated because in the absence of gene N product, over half of the mRNA synthesis initiated at PR terminates at tRl , and in the
173 presence of gene N product mRNA synthesis initiated at PR continues through gene Q. However, }`c17 contains a new promoter located to the right of tR1. Genes O and P, but not Q, are transcribed from this promoter in the absence of gene N product. In the presence of gene N product, gene Q is transcribed (Couturier et al., 1973), suggesting the presence of an N-sensitive transcription terminator between P and Q, tR2 (Fiandt et al., 1971). nin5 is postulated to delete tR2. A secondN-independent variant is }`cl 7byp in which the c17 mutation allows expression of genes O and P and byp allows increased Q expression (Hopkins, 1970; Butler and Echols, 1970). Since byp maps to the right of the nin5 region, byp and nin5 cannot be both interpreted as eliminating tR2. ren is a gene which protects }` from a complex exclusion phenomenon by rex, a gene mapping in the }` immunity region. The ren20 and ren51 mutations lie to the right of gene P and to the left of nin5. nin5 phages however, are ren-, indicating that nin5 removes the right portion of the ren gene (Toothman and Herskowitz, 1980). puq3 and puql 6 are poorly understood mutations which also map within the nin5 region (Sternberg and Enquist, 1979). These mutations allow increased synthesis of the gene Q product under conditions where synthesis of gene Q product is limited, pasB is a mutation lying within the nin5 regions which allows a XRed- to grow on a polA- bacterial strain (Shizuya et al., 1974). It is not clear whether pasB inactivates an enzymatic function or is a control mutation, nin5 also has the pasB phenotype as might be expected ifpasB defines a gene. We have previously reported that an insertion of Tn903 which inactivates gene S is located 3310 bp from the right end of }` DNA (Chiang et al., 1979), corresponding to position 93.1%}`. The location determined from BamHI and HindlII is also 93.1%}`, but the transposon lies to the right of the EcoRI cleavage site at 93.1%}` (R. Young, personal communication). We assign the ribosome binding site at 3410 bases from the right end to gene S on this basis. A ribosome binding site to the left of gene S, apparently located in or near the 6S RNA sequence, was found in both this study and the previous study (Chiang et al., 1979). This ribosome binding site might or might not have any biological significance. There are two possible binding sites within the 6S
sequence (Sklar et al., 1975), each containing an ATG and a sequence complementary to the 3'-end of 16S rRNA (Shine and Dalgarno, 1974). One of these is located near the beginning of the 6S sequence and contains a 5 bp Shine and Delgarno sequence separated by six bp from the ATG codon. The second contains a 5 bp complementary sequence separated by 8 bp from the ATG codon. Both ATG codons occur in the same reading frame and neither sequence contains a terminator between the ATG codon and the end of the 6S RNA molecule. A polypeptide synthesized using the first ATG codon could contain 53 amino acids encoded in the 6S RNA, and a polypeptide synthesized Using the second ATG codon could contain 21 amino acids encoded in the 6S RNA. If the 6S RNA were extended by Q-dependent antitermination, an additional eleven amino acids could be incorporated before a polypeptide termination codon is reached (D. Daniels, personal communication). Since the 6S RNA is presumably synthesized constitutively, the absence of a polypeptide termination codon could provide a convenient mechanism to prevent extensive translation of the 6S RNA since the ribosome reaching the end of the 6S RNA would not readily dissociate and by remaining bound would prevent polypeptide synthesis by additional ribosomes. In addition, the polypeptide product might not be active in the absence of the carboxyl terminal amino acids not encoded by the 6S RNA. We have located eleven ribosome binding sites to the left of the transposon insertion site in },pk3 DNA. We tentatively identify the site located immediately to the left of the }`pk3 transposon insertion site as gene Q. Echols and Murialdo (1978) and Szybalski and Szybalski (1979) place gene Q between map coordinates 90.8-92.1 or 90.6-92 %}`, respectively. This location is based in part on the finding that the galM3 substitution which terminates at 91.7 %}` removes }` DNA corresponding to genetic markers Qam72, Qam21, and Qamll7, but does not remove DNA corresponding to Qam57 (Fiandt et al., 1971). This indicates that Q extends on both sides of position 91.7 %}`. In add.ition, the PQA4 deletion extends to position 90.5 %}` (Sternberg et al., 1979; see also Szybalski and Szybalski, 1979). These considerations closely fix the beginning of gene Q to about position 90.6 %}, which would place it about 4500 bp from the right end of }`, assuming 48 000 bp for }`. Some uncertainty about the identification of this ribosome
174 binding site with gene Q exists since ;kpk3 is viable and yet contains a transposon inserted about 510 bp from the ribosome binding site. If this measurement is correct and if the estimate of 620 bp for the length of gene Q is correct, based on a polypeptide molecular weight of 23 000, it would appear that the transposon may be inserted near the end of the Q gene. Possibly the gene Q polypeptide could be active despite having an altered carboxyl terminus or the gene Q polypeptide may be inactive. If the Q polypeptide has been inactivated by the insertion of the transposon, the phage should not be viable unless the transposon provides a new rightward promoter for ~ late genes or renders the phage Q-independent in some other way. Alternatively, this ribosome-binding site may not correspond to the polypeptide-initiation site for gene Q. Another ribosome binding site apparently present on both Xpk3 and Xnin5dk6 DNAs lies about 4750 bases from the right end of ~. In this region, between nin5 and Q lie two known mutations, byp and qinlO1. qinlO1 is a promoter mutation which allows constitutive expressive of gene Q and the ~ late genes (Dambly et al., 1979). byp might also be a new promoter (Mark, 1973). Probably neither byp or qinlO1 achieve their effect by inactivating a gene product. Seven ribosome binding sites fall within the region of DNA we assign to nin5. Our measurements for nin5 based on heteroduplex mapping place the right terminus ofnin5 about 5200 bp from the right end of X DNA. The ribosome binding sites located 5060 bases from the right end of ~ DNA probably is not removed by the nin5 deletion. Since the nin5 deletion (5.8 %X units) removes about 2800 bp, assuming 48000 bp for ~ (Szybalski et al., 1977), the left end of nin5 would fall about 8000 bp from the right end of ;~ DNA. Thus, the nin5 deletion would be expected to remove the ribosome binding site located 7500 bases from the right end, and not to remove the ribosome-binding site located 8190 bases from the right end. The extent of the nin5 deletion is shown in Fig. 3 assuming nin5 is located between 5200 and 8000 bp from the right end of ~ DNA. A total of seven ribosome-binding sites would be removed by this deletion. If a gene is defined by the ribosome-binding site 8190 bases from the right end of ~ it would probably also be inactivated. Two ribosome binding sites
between the right end of nin5 and the gene presumed to be Q would not be removed by the nin5 deletion. This assignment is consistent with the locations of ribosome-binding sites on ~nin5dk6. The ribosomebinding sites on the two DNA molecules can be aligned by assuming that the complex of ribosome binding sites observed between 9 1 0 0 - 1 0 0 0 0 bases from the right end of kpk3 corresponds to the complex of ribosome-binding sites located 6300-7200 bases from the right end of knin5dk6. The difference between these values, 2800 bases, corresponds to the length of the nin5 deletion. If the bound ribosome on knin5dk6 located between 5000 and 5500 bases from the right end of DNA are assumed to define a single ribosome-binding site lying to the left of nin5, this ribosome-binding site would probably correspond to the ribosomebinding site located on kpk3 8190 bases from the right end of ~ DNA. The profile of the bound ribosomes at this position however suggest that on knin5dk6 there may be two very close ribosomebinding sites. These could be two ribosome-binding sites brought close together by the nin5 deletion. If so, the other ribosome-binding site is likely to be the one located on kpk3 5060 bases from the right end of k DNA. Other possible locations for the nin5 deletion that are consistent with the data for the locations of ribosome-binding sites on kpk3 and knin5dk6 fit the measured location of nin5 less well. In summary, it appears that there are about 20 ribosome-binding sites located to the right of genes O and P. A ribosome-binding site which may correspond to ren lies about 200 bases left of the left end point of nin5. The nin5 deletion would probably remove coding sequences for a gene associated with this ribosome-binding site, as was found for ten (Toothman and Herskowitz, 1980). Seven ribosomebinding sites are removed by the nin5 deletion. Two ribosome-binding sites may exist between the right end point of nin5 and gene Q. Genes in this region may be non-essential since the PQA4 deletion extends nearly to gene Q (Sternberg et al., 1979). A ribosomebinding site has been tentatively assigned to gene Q and another to the 6S region of ~,. Ribosome-binding sites likely to correspond to gene S and Rz have been located near transposons which inactivate these genes. A ribosome-binding site which may correspond to gene R lies between the sites assigned to genes S and Rz. Beyond gene Rz are four or five ribosome-binding
175 sites which may indicate the presence of up to five new ~ genes transcribed as part of the late mRNA.
ACKNOWLEDGEMENTS We thank Ryland Young for his phage strains and for helpful advice during the course of this work. We thank
Charles
Roessner
and
Leslie Merritt
for
assistance with ribosome and tRNA purification and for assistance with the electron microscopy. This work was supported by the National Institute of Health (grant R01 GM 24432).
REFERENCES Alberts, B. and Herrick, G.: DNA-cellulose chromatography. Methods Enzymol. 21 (1971) 198-214. Buffer, B. and Echols, H.: Regulation of bacteriophage h development by gene N: properties of a mutation that bypasses N control of late protein synthesis. Virology 40 (1970) 212-222. Calame, K. and Ihler, G.: Visualization of ribosome-singlestranded DNA complexes in the electron microscope. Biochemistry 16 (1977a) 964-971. Calame, K. and lhler, G.: Physical location of ribosome binding sites on lambda DNA. J. Mol. Biol. 116 (1977b) 841-853. Chiang, T., Roessner, C. and lhler, G.: Location of ribosome binding sites on the right end of lambda DNA. J. Mol. Biol. 135 (1979) 893-906. Court, D. and Sato, K.: Studies of novel transducing variants of lambda: dispensibility of genes N and Q. V'trology 39 (1969) 348-352. Couturier, M., Dambly, C. and Thomas, R.: Control of development in temperate bacteriophage, V. Sequential activation of the viral functions. Mol. Gen. Genet. 713 (1973) 244-250. Dambly, C. Delstanche, M. and Gathoye, A.M.: qin 101: promoter mutation which allows the constitutive expression of the late genes. J. Virol. 30 (1979) 14-20. Echols, H. and Murialdo, H.: Genetic map of bacteriophage lambda. Microbiol. Rev. 42 (1978) 577-591. Fiandt, M., Hradecna, Z., Lozeron, H.A. and Szybalski, W.: Electron micrographic mapping of deletions, insertions, inversions, and homologies in the DNAs of coliphages lambda and phi80, in Hershey, A.D. (Ed.), The Bacteriophage Lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1971, pp. 329-354. Hendrix, R.: Identification of proteins coded in phage lambda, in Hershey, A.D. (Ed.), The Bacteriophage Lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971, pp. 355±370.
Hopkins, N.: Bypassing a positive regulator: isolation of a x mutant that does not require N product to grow. Virology 40 (1970) 223-229. Ihler, G. and Nakada, D.: Selective binding of ribosomes to initiation sites on single-stranded DNA from bacterial viruses. Nature 228 (1970) 239-242. Ihler, G. Calame, K. and Nakada, D.: Electron microscopic mapping of ribosome-binding sites on single-stranded DNA. Methods Enzymol. 59 (1979) 837-851. Lewin, B. Gene Expression, Vol. 3, Plasmids and Phages. Wiley, New York, 1977, pp. 274-535. Mark, K.-K.: Effect of the N-bypass mutations nin and byp on the rightward transcription in coliphage h. Mol. Gen. Genet. 124 (1973) 291-304. Pagano, T.S. and Hutchinson, C.A.: Small, circular, viral DNA: Preparation and analysis of SV40 and ~X174 DNA. Methods Virol. 5 (1971) 79-123. Scherer, G.: Nucleotide sequence of the O gene and of the origin of replication in bacteriophage lambda DNA. Nucl. Acids Res. 5 (1978) 3141-3156. Schwarz, E., Sherer, G., Hobom, G. and KOssel, H.: Nudeotide sequence of cro, cII, and part of the O gene in phage h DNA. Nature, 272 (1978) 410-414. Shine, J. and Dalgarno, L.: The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71 (1974) 1342-1346. Shizuya, H., Brown, D. and Campbell, A.: DNA polymerase I-independent mutants of coliphage lambda. J. Virol. 13 (1974) 947-952. Sklar, J., Yot, P. and Weissman, S.M. : Determination of genes, restriction sites, and DNA sequences surrounding the 6S RNA template of bacteriophage lambda. Proc. Natl. Acad. Sci. USA 72 (1975) 1817-1821. Sternberg, N. and Enqulst, L.: Analysis of coliphage lambda mutations that affect Q gene activity: puq, byp, and nin5. J. Virol. 30 (1979) 1-13. Sternberg, N., Hamilton, D., Enquist, L. and Weisberg, R.: A simple technique for the isolation of deletion mutants of phage lambda. Gene 8 (1979) 35 -51. Szybalski, E.H., Honigman, A., Rosenvold, E.C., Fiandt, M. and Szybalski, W.: A deletion in the P-Q (ninR) region of phage hb2imm21 conferring partial gene N21 independence. Gene 2 (1977) 295-297. Szybalski, E.H. and Szybalski, W. A comprehensive molecular map of bacteriophage h, Gene 7 (1979) 217- 270. Toothman, P. and Herskowitz, I. Rex-dependent exclusion of lambdoid phages. Virology (1980) (in the press). Young, R., Way, J., Way, S., Yin, J. and Syvanen, M.: Transposition mutagenesis of bacteriophage lambda: a new gene affecting cell lysis. J. Mol. Biol. 132 (1979) 307322. Westmoreland, B.C., Szybalski, W. and Ris, H., Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy, Science, 163 (1969) 1343-1348. Communicated by W. Szybalski.
167
L o c a t i o n s o f r i b o s o m e - b i n d i n g sites in the n i n 5 region o f b a c t e r i o p h a g e X (E. coli; deletion; single-stranded DNA; mRNA; electron microscopy; transposons; restriction enzymes)
Tom Chiang and Garrett lhler Department of Medical Biochemistry, Texas A & M College of Medicine, College Station, TX 77843 (U.S.A.)
(Received November 16th, 1979) (Revision received and accepted February 26th, 1980)
SUMMARY Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region. These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene R z and another inserted near gene Q. These ribosome binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible X genes.
INTRODUCTION Calame and Ihler (1977a) developed an electron microscopic procedure for locating ribosome binding sites on DNA. Ribosomes are allowed to bind to single-stranded DNA serving as an analog of mRNA and the DNA-ribosome complexes are visualized using the electron microscope. The positions of the bound ribosomes are determined relative to a reference point such as a transposon insertion site or an end created by a restriction enzyme. Most of the ribosome binding sites detected by this procedure may correspond to polypeptide initiation sites used in vivo since binding on X DNA (Calame and Ihler, 1977a,b) or T7 DNA (Ihler and Nakada, 1970) occurs predominantly to that strand of DNA which contains the same base sequence as mRNA. This procedure may be especially useful for Abbreviations: bp, base pairs; %h unit, 480 bp;p, promoter; s, startpoint; t, terminator.
studying regions of DNA for which there is no genetic detection system available. For example, there are several extensive regions in the bacteriophage X genome which are so far devoid of known genes despite the fact that X has been extensively studied by most of the techniques of genetics and molecular biology. One of these regions is the b2 region, defined by the b2 deletion, which is known to code for polypeptides of unknown function (Hendrix, 1971). Another is the region defined by the nin5 deletion and a third is the region between gene R z and the right end of X DNA. We have been locating ribosome-binding sites on bacteriophage X DNA and have now located the ribosome-binding sites on the right-hand 40% of the DNA (Calame and Ihler, 1977b; Chiang et all, 1979). Comparison of the locations of these ribosome-binding sites with the known genetic and physical map of and with the limited amount of DNA sequence data presently available for X DNA, shows a general correlation between the locations of some ribosome binding
168 sites and the locations of polypeptide initiation sites for known genes. Other ribosome-binding sites have been found in genetically silent regions and these may indicate the existence of as yet unknown genes, although definitive proof that these ribosome-binding sites are functional in vivo must await detection of the polypeptides. We have now located nearly forty ribosome-binding sites on the right-hand 40% of DNA, of which sixteen may correspond to known genes.
(c) Preparation of phage T4 gene 32 protein Bacteriophage T4 gene 32 protein was purified from a T4-infected E. coli cell lysate on a denatured calf thymus DNA-cellulose column as described (Alberts and Herrick, 1971; Chiang et al., 1979). Gene 32 protein prevents random base interactions of DNA which would otherwise be fixed by glutaraldehyde and also greatly improves the morphology of single-stranded DNA under electron microscope. (d) Binding of ribosomes to single-stranded DNA
MATERIALS AND METHODS
(a) Bacteria and phage ~in5dk6 phage (~519cI857nin5Sam7Rz: :Tn903) contains the nin5 deletion and a transposable DNA element inserted into gene Rz (Young et al., 1979). Xpk3 (~b519ci857Tn903 Sam7) has the transposon inserted about 3900 bp from the right end. ~pk35nin5 (~b519b515ci857cli: :Tn903nin5Sam7) contains the transposon inserted in gene clI. Phage lysogens, obtained from R. Young (Young et al., 1979), were grown in broth at 30°C to between 5 × 10 a cells/ml to 109 cells/ml before they were thermoinduced for 15 min at 43°C. After an additional 2 - 3 h incubation at 37°C, cells were concentrated by centrifugation and lysed by rapid freezing and thawing. Phage particles were purified by standard CsC1 density gradient centrifugation. ¢X174 single-stranded DNA was obtained by phenol extraction of purified ~bX174 am3 phage by the method of Pagano and Hutchinson (1971). E. coli ribosomes, initiation factors and charging enzymes were prepared from strain 238 (DNase I-, B-) as described by Ihler et al. (1979).
Ribosome-binding reactions were carried out and the DNA-ribosome complexes were purified as described previously (Calame and Ihler, 1977a).
(e) Electron microscopy of DNA-ribosome complexes and of DNA heteroduplexes The DNA-ribosome complexes were fixed, spread and picked up on carbon-coated grids as described by Ihler et al. (1979). DNA molecules with a transposon (containing a stem and loop) and one or more bound ribosomes were photographed. Distances were determined by tracing the projected negative using a Numonics Graphics Calculator (Model 1224, Numonics Corporation). One or more ¢X174 single-stranded DNA molecules in the same field were also measured and used as a length standard. DNA heteroduplexes were prepared and measured by the formamide technique of Westmoreland et al. (1969) as described previously (Chiang et al., 1979).
RESULTS
(b) Preparation of phage DNA for ribosome binding Single-stranded ~,pk3 DNA was prepared from purified phage particles by simultaneous lysis and denaturation in 0.2 N NaOH. The denatured DNA (about 50/ag/ml) was neutralized and dialysed at 4°C against 10 mM Tris, pH 7.4 and 1 mM magnesium acetate. In order to assure complete annealing of the inverted repeat of the transposon, the singlestranded DNA was incubated for 2 rain at 65°C before use in the binding reaction.
In a previous study we determined the number and locations of the ribosome binding sites on the right end of k DNA (Chiang et al., 1979). Those studies utilized knin5dk6, which carries the nin5 deletion, and so we employed another phage, kpk3, which is nin + to determine the number and locations of ribosome binding sites within the nin5 region. Measurements on kninSdk6 DNA were made relative to a transposon insertion site in gene R z (Young et al., 1979). kpk3 contains the same transposon inserted at a different
169
site. To ensure that the locations of ribosome binding sites determined using one transposon as a reference point would correspond closely with the locations determined using the other transposon, we measured the positions cf both transposon insertion sites simultaneously by heteroduplexing DNA from ~.pk3 with DNA from Xnin5dk6. We found the transposon in Xnin5dk6 to be inserted 2310 + 90 bp from the right end of the X DNA (when measuring the heteroduplex molecule and using double-stranded ¢X174 as the length standard), and 2340-+ 330 bases from the right end, when measuring single-stranded Xnin5dk6 DNA using single-stranded ¢X174 as the length standard. This position is in close agreement with the location determined by Young et al. (1979). The transposon insertion site in Xpk3 DNA is located 1590 -+ 170 bp left of the transposon in ?,nin5dk6, corresponding to 3900 -+ 180 bp from the right end of ?~DNA. Assuming 48000 bp for ~t DNA, this corresponds to map position 91.9 %~, which differs significantly from the position of 92.3 %~ reported by Young et al. (1977). Using heteroduplexes between ?~nin5dk6 and ]tpk3, we located the right terminus of nin5 about 5160 -+ 250 bp from the right end of ?t DNA (Chiang et al., 1979). We have now made heteroduplexes between ?~pk3 and ~pk35nin5. For a length standard we use 3900 bp as the distance from the right end of ~ DNA to the Xpk3 transposon insertion site. The transposon insertion sites in Xpk3 and ?~pk35nin5 are separated by 3470 ± 180bp. The right terminus of nin5 is located 1360 + 90 bp from the transposon insertion site in ;kpk3 which corresponds to 5260 ± 270 bp from the right end of X DNA, in close agreement with
pk35
the previous determination of 5160 bp. The left terminus of nin5 is 2110 bp from the transposon in
?tpk35nin5. The nin5 deletion was originally measured to be a 5.4 %~ deletion. More recently Szybalski et al. (1977) have reported nin5 to be a 5.8% deletion with its right end at 89.5 %h, i.e., 5040 bp from the right terminus. Assuming 48 000 bp for ~, DNA (Szybalski et al., 1977), the length of nin5 would be about 2800 bp. We will take the endpoints of nin5 to be located 5200 and 8000 bp from the right end of ~t DNA. The transposon insertion site in ~pk35nin5 is within gene clI, 180 bp from the clI polypeptide initiation site (D. Moore, personal communication). Taking 2800 bp as the length of nin5, this would place the clI polypeptide initiation sites 10350 bp from the right end of ?~ and the gene O polypeptide initiation site about 10000 bp from the right end of ~, DNA. Fig. 1 shows the separation in bp from these various markers. Fig. 2 shows a histogram of the number of ribosomes bound in each 100 base interval for ~v~in5dk6 and ?tpk3. Table I lists the number of bases separating the ribosome binding sites from the transposon insertion sites on ?~pk3 and ?,nin5dk6 and the locations of the ribosome binding sites in bases from the right end of ?t DNA. It can be seen that the measured locations correspond closely for the ribosome binding sites common to the two ~ DNA molecules. For example, measurements on both ~ i n 5 d k 6 and ~.pk3 DNA demonstrate the presence of a ribosome binding site located 4750 bases from the right end of ~. Another ribosome binding site is located 4410 bases from the
dk6
pk3
?
I
nin5
I
1260
2110
.ia "I-
--2000
"-)"
?
?
3',~
1590
"J"
2310
1360
Fig. 1. Distance measurements. The measurements of the locations of the three transposons referred to in the text arid of the right and left ends of the nin5 deletion are summarized here. Two separate measurements for the location of the right end of nin5 were made. The first measurement, made using heteroduplexes between hpk3 and ~ninSdk6, gave a value of 5160 + 250 bp from the right end of h DNA. Double-stranded OX174 (5386 bp) was used as a length standard to determine the position of the transposon in knin5dk6 relative to the right end. A second measurement for the location of the right end of nin5 gave a value of 5260 + 270 bp from the right end of h. This measurement was made using heteroduplexes between kpk3 and hpk35nin5. The previously determined distance of 3900 bp from the right end for the transposon in hpk3 was used as the length standard.
170 TABLE I Location of ribosome binding sites on hpk3 and hninSdk6. The distance of ribosome binding sites from the transposon in hnin5dk6 or hpk3 is given in bases measured using ~X174 DNA (5386 bases) as a length standard. The distance from the right end is calculated assuming that the transposon insertion site in hnin5dk6 is located 2310 bases from the right end and that the transposon-insertion site in hpk3 is located 3900 bases from the right end. Distances for ribosome binding sites on hnin5dk6 are corrected for the nin5 deletion (2800 bases) where appropriate. For the site which could lie on either or both sites of nin5, it was assumed that it lies on both sides and these are the values given (5150, 8150) in the data for hnin5dk6
hnin5dk6
hpk3 Bases from right end
Bases from transposofi
3700 4410 4750 5060 5620 5950 6200 6550 6750 7060 7500 8190 9100-10000
Number of ribosomes
200 510 850 1160 1720 2050 2300 2650 2850 3160 3600 4290 5200-6100
3 5 5 5 2 5 11 I1 8 5 6 2 13
Polypeptide product
Bases from right end
Bases from transposon
Number of ribosomes
220 1165 1520 1980 2590 3010 3410 3670 4260 4770 5150
2090 1145 790 330 280 700 1100 1360 1950 2460 -2900
3 3 8 6 9 3 8 8 9 7 12
8150 9100-10000
-2900 4000-4900
12 20
9000 35000 13000 17000 23000 16000 15000 9600 22000 19000 13000 21000 12000 9200 11000 7400 12000 16000 26000 -
nin5
!
,k6
,"I, 4 g~ end
|
,
nin5
J
6q
,,
I I
p.k3
2 E)
9
8
7
6
5
4
5
2
I
0 end
KI LOBASES Fig. 2. Histogram of bound rbosomes. The locations of bound ribosomes on hpk3 DNA and ~inSdk6 DNA were measured relative to the transposons using ~X174 as a length standard. All ribosomes falling into each 100 base increment were pooled for plotting the histogIams. The two histograms correspond for sites located 3700, 4]40, 4750, and 5060 basesfrom the fight end of h. Ribosomes bound to hpk3 DNA between 0 and 3500 bases from the right end of DNA are omitted due to the small number of ribosomes measured in this region. The measured location for the fight end of nin5 is 5200 bases from the fight end of h DNA. nin5 deletes about 2800 bp from hnin5dk6 (upper histogram). This DNA is present in ;~pk3 (lower histogram) in the region located between 5200 and 8000 bases from the rght end of h DNA.
171 right end using ),pk3 DNA and 4260 bases from the right end using Xnin5dk6 DNA. This ribosome binding site may correspond to the polypeptide initiation site for gene Q. We have not a~tempted to extensively locate ribosome-binding sites on Xpk3 tO the right of the transposon since this region has been studied previously (Chiang et al., 1979) but those ribosomes which we have measured fall near ribosome-binding sites found previously except for one ribosome bound about 730 bases from the right end of X. We have confirmed, using Xpk3, the location of a ribosome binding site previously found with Xnin5dk6 in or very near the region from which 6S RNA is transcribed. Within the 6S RNA sequence itself are only 3 AUG codons, each of which has a preceeding sequence which might function as a Shine and Dalgarno (1974) sequence.
right end of )`. The distances separating these ribosome binding sites range from 200 to 565 bases, corresponding to potential polypeptides ranging in molecular weight from 7500 to 21000 if it is assumed ribosome binding sites all correspond to polypeptide initiation sites for different genes (Table I). Since the right terminus of nin5 has been measured as 5160 -+ 250 bp or 5260 -+ 270 bp, it is likely but not certain that the ribosome binding site at 5060 bases from the right end of ), DNA lies to the right of nin5. However, it is certain that the remaining seven ribosome binding sites located between 5620 and 7500 bases must be removed by the nin5 deletion. Assuming 2800 bp for the length of the deletion and placing the right terminus at 5200 bp from the right end of), DNA, the left end ofnin5 would lie 8000 bp from the right end of ), DNA which is well beyond the ribosome binding site located 7500 bases from the right end of X DNA. To confirm this, we have compared the location of ribosome binding sites on )`pk3 and ),ninSdk6 (Fig. 2). To the left of the transposon on )`pk3 are eleven ribosome binding sites. In addition a cluster of perhaps four ribosome binding sites is found between 9100 and 10000 bases from the right end of X. This cluster must lie well to the left of the nin5 region. A similar cluster of ribosome binding sites therefore should be found on ),nin5dk6. Such a cluster can be found located 6300-7200 bases from the right end of Xnin5dk6 or 9 1 0 0 - 1 0 0 0 0 bases from the right end of ), if 2800 bases are added to compensate for the nin5 deletion. We are attempting to measure the location of the ribosome binding sites more exactly by binding ribosomes to ),pk35nin5 DNA, which has
5'-TGGGUAAAUUUGACUCAACGAUG-3' 5'-UGGUAGUGAGAUG-3' 5'-AGAGGUCUGCAAAAUG-3' The AUG codon in the first sequence is followed one codon later by the polypeptide termination codon UAA. The other two are read in the same phase and no polypeptide termination codons occur up to the end of the 6S RNA sequence. Either of these latter two sequences could correspond to the observed ribosome binding site. The second sequence would code for the longer polypeptide. The third sequence has a more convincing Shine and Dalgarno sequence, but would code for a much shorter polypeptide. Eight ribosome binding sites have been located on ),pk3 DNA between 5060 and 7500 bases from the
[
J
•
1
nin~
I
•
1
1
1
t~
1
l
1
1
1 , I
,o
I erd
KILOBASES from RIGHT END
Fig. 3. Location of ribosome binding sites. The stem and loop symbols indicate the sites of insertion of two transposons, one in Xnin5dk6 (Rz, 2310 bases from the right end) and one in hpk3 (3900 bases from the right end). Arrows indicate the locations of ribosome binding sites measured in kilobases from the right end of ~, DNA. Arrows in the upper row indicate the location of ribosome binding sites determined using hnin5dk6 DNA, assuming the right and left ends ofnin5 are located 5200 and 8000 bp respectively from the right end of h DNA. Arrows in the lower row indicate the locations of ribosome binding sites determined using hpk3 DNA. Ribosome binding sites on kpk3 between 6S RNA and the fight end of h were not determined. Solid triangles indicate the endpoints of nin5 (5040 and 7823 bp from the right end) as determined by Szybaiski et al. (1977) (see also Szy-
balski and Szybalski, 1979).
172 a transposon inserted in clI very near the polypeptide initiation site for gene O. Fig. 3 shows the position of ribosome binding sites that must lie outside the nin5 region, either to the right or to the left. The only potential ambiguity lies with deciding whether the ribosome binding site located on kpk3 DNA 5060 bp from the right end is or is not removed by the nin5 deletion. The interpretation which we prefer is that the ribosome binding site at 5060 bases is not removed by nin5. The profile of bound ribosomes on )~nin5dk6 between 5000 and 5500 bases from the right end suggests the presence of two close ribosome binding sites. If so, one of these ribosome binding sites is likely to correspond to the ribosome binding site found on ~pk3 5060 bases from the right end of ~. The other is likely to correspond to the ribosome binding site found on ;~pk3 at 8190 bases from the right end of k. On ~nin5dk6 these two ribosome-binding sites would be brought to within 350 bases of each other by the 2800 base nin5 deletion. If it is assumed that the profile of bound ribosomes located 5300 bases from the right end of ~nin5dk6 DNA represents a single ribosome-binding site, this site could correspond either to the site on kpk3 5060 bases from the right end or to the site 8190 bases from the right end. In the former case the end points of nin5 would have to be about 5400 and 8200 bases from the right end of ~. DNA. In the latter case the end points of nin5 would be about 5000 and 7800 bases from the right end. The ribosome binding site located 4770 bases from the right end of ;~nin5dk6 would align equally well with Xpk3 at this position or at a position 7550 bases from the right end. The latter assignment however seems unlikely since it would require that nin5 remove the ribosome binding site located on Xpk3 at 4750 bases from the right end. This appears to be too far from the measured location of the right terminus of nin5 at 5160 + 250 or 5260 + 270 bp from the right end, and even from the 5040 bp location measured by Szybalski et al. (1977).
DISCUSSION In the absence of the X repressor protein, rightward and leftward mRNA synthesis begins on ~ DNA
at two promoters, PL and PR, which are located on opposite sides of the immunity region containing the gene (cI) that codes for the X repressor. The rightward mRNA transcript terminates at the tR~ and tR2 sites located after genes cro and P respectively. The product of the N gene, contained in the leftward mRNA transcript, acts as an anti-termination factor for RNA synthesis, probably by binding to RNA polymerase, and allows mRNA transcription to proceed through tR1 and tR2 to include genes cII, O, P, the nin5 region and gene Q. Gene cII plays a role in the establishment of lysogeny. Genes O and Pare required for X DNA replication. Gene Q codes for a protein which is required for expression of all late X genes and acts at a site termed P'R (or pQ) located to the right of Q. A small RNA molecule, 6S RNA, is synthesized from the region to the right of Q and it has been proposed that the Q protein may act as an antiterminator for the 6S molecule, allowing rightward RNA synthesis to continue from P'R to genes S, R, and Rz, which are required for lysis of the bacterial host, and then to genes coding for phage structural proteins. A general discussion of the molecular biology of bacteriophage ~ can be found in Lewin (1977). The first three genes transcribed from PR, cro, cII, and O, have been sequenced. The polypeptide initiation site for cro is 19 bp from s R and the cro gene is 201 bp long, There are 118 bp between the cro polypeptide site and the polypeptide initiation site of gene cII. cII is 294 bp long (Schwarz et al., 1978) and is separated by 32 bp from the presumed polypeptide initiation site of gene O. Gene O is 897 bp long (Scherer, 1978). The region between genes P and Q is largely devoid of known genes and no polypeptides have been identified which are coded for by genes in this region. Most or all of the genes in this region must be dispensible for normal ~ development since the nin5 deletion removes about 2800 bp between P and Q without affecting viability. The nin5 deletion was isolated as a ~ variant which could grow in the absence of gene N product (Court and Sato, 1969). Apparently an N-sensitive terminator(s) for mRNA synthesis exist(s) in this region, between P and Q. The existence of this terminator cannot be readily demonstrated because in the absence of gene N product, over half of the mRNA synthesis initiated at PR terminates at tRl , and in the
173 presence of gene N product mRNA synthesis initiated at PR continues through gene Q. However, }`c17 contains a new promoter located to the right of tR1. Genes O and P, but not Q, are transcribed from this promoter in the absence of gene N product. In the presence of gene N product, gene Q is transcribed (Couturier et al., 1973), suggesting the presence of an N-sensitive transcription terminator between P and Q, tR2 (Fiandt et al., 1971). nin5 is postulated to delete tR2. A secondN-independent variant is }`cl 7byp in which the c17 mutation allows expression of genes O and P and byp allows increased Q expression (Hopkins, 1970; Butler and Echols, 1970). Since byp maps to the right of the nin5 region, byp and nin5 cannot be both interpreted as eliminating tR2. ren is a gene which protects }` from a complex exclusion phenomenon by rex, a gene mapping in the }` immunity region. The ren20 and ren51 mutations lie to the right of gene P and to the left of nin5. nin5 phages however, are ren-, indicating that nin5 removes the right portion of the ren gene (Toothman and Herskowitz, 1980). puq3 and puql 6 are poorly understood mutations which also map within the nin5 region (Sternberg and Enquist, 1979). These mutations allow increased synthesis of the gene Q product under conditions where synthesis of gene Q product is limited, pasB is a mutation lying within the nin5 regions which allows a XRed- to grow on a polA- bacterial strain (Shizuya et al., 1974). It is not clear whether pasB inactivates an enzymatic function or is a control mutation, nin5 also has the pasB phenotype as might be expected ifpasB defines a gene. We have previously reported that an insertion of Tn903 which inactivates gene S is located 3310 bp from the right end of }` DNA (Chiang et al., 1979), corresponding to position 93.1%}`. The location determined from BamHI and HindlII is also 93.1%}`, but the transposon lies to the right of the EcoRI cleavage site at 93.1%}` (R. Young, personal communication). We assign the ribosome binding site at 3410 bases from the right end to gene S on this basis. A ribosome binding site to the left of gene S, apparently located in or near the 6S RNA sequence, was found in both this study and the previous study (Chiang et al., 1979). This ribosome binding site might or might not have any biological significance. There are two possible binding sites within the 6S
sequence (Sklar et al., 1975), each containing an ATG and a sequence complementary to the 3'-end of 16S rRNA (Shine and Dalgarno, 1974). One of these is located near the beginning of the 6S sequence and contains a 5 bp Shine and Delgarno sequence separated by six bp from the ATG codon. The second contains a 5 bp complementary sequence separated by 8 bp from the ATG codon. Both ATG codons occur in the same reading frame and neither sequence contains a terminator between the ATG codon and the end of the 6S RNA molecule. A polypeptide synthesized using the first ATG codon could contain 53 amino acids encoded in the 6S RNA, and a polypeptide synthesized Using the second ATG codon could contain 21 amino acids encoded in the 6S RNA. If the 6S RNA were extended by Q-dependent antitermination, an additional eleven amino acids could be incorporated before a polypeptide termination codon is reached (D. Daniels, personal communication). Since the 6S RNA is presumably synthesized constitutively, the absence of a polypeptide termination codon could provide a convenient mechanism to prevent extensive translation of the 6S RNA since the ribosome reaching the end of the 6S RNA would not readily dissociate and by remaining bound would prevent polypeptide synthesis by additional ribosomes. In addition, the polypeptide product might not be active in the absence of the carboxyl terminal amino acids not encoded by the 6S RNA. We have located eleven ribosome binding sites to the left of the transposon insertion site in },pk3 DNA. We tentatively identify the site located immediately to the left of the }`pk3 transposon insertion site as gene Q. Echols and Murialdo (1978) and Szybalski and Szybalski (1979) place gene Q between map coordinates 90.8-92.1 or 90.6-92 %}`, respectively. This location is based in part on the finding that the galM3 substitution which terminates at 91.7 %}` removes }` DNA corresponding to genetic markers Qam72, Qam21, and Qamll7, but does not remove DNA corresponding to Qam57 (Fiandt et al., 1971). This indicates that Q extends on both sides of position 91.7 %}`. In add.ition, the PQA4 deletion extends to position 90.5 %}` (Sternberg et al., 1979; see also Szybalski and Szybalski, 1979). These considerations closely fix the beginning of gene Q to about position 90.6 %}, which would place it about 4500 bp from the right end of }`, assuming 48 000 bp for }`. Some uncertainty about the identification of this ribosome
174 binding site with gene Q exists since ;kpk3 is viable and yet contains a transposon inserted about 510 bp from the ribosome binding site. If this measurement is correct and if the estimate of 620 bp for the length of gene Q is correct, based on a polypeptide molecular weight of 23 000, it would appear that the transposon may be inserted near the end of the Q gene. Possibly the gene Q polypeptide could be active despite having an altered carboxyl terminus or the gene Q polypeptide may be inactive. If the Q polypeptide has been inactivated by the insertion of the transposon, the phage should not be viable unless the transposon provides a new rightward promoter for ~ late genes or renders the phage Q-independent in some other way. Alternatively, this ribosome-binding site may not correspond to the polypeptide-initiation site for gene Q. Another ribosome binding site apparently present on both Xpk3 and Xnin5dk6 DNAs lies about 4750 bases from the right end of ~. In this region, between nin5 and Q lie two known mutations, byp and qinlO1. qinlO1 is a promoter mutation which allows constitutive expressive of gene Q and the ~ late genes (Dambly et al., 1979). byp might also be a new promoter (Mark, 1973). Probably neither byp or qinlO1 achieve their effect by inactivating a gene product. Seven ribosome binding sites fall within the region of DNA we assign to nin5. Our measurements for nin5 based on heteroduplex mapping place the right terminus ofnin5 about 5200 bp from the right end of X DNA. The ribosome binding sites located 5060 bases from the right end of ~ DNA probably is not removed by the nin5 deletion. Since the nin5 deletion (5.8 %X units) removes about 2800 bp, assuming 48000 bp for ~ (Szybalski et al., 1977), the left end of nin5 would fall about 8000 bp from the right end of ;~ DNA. Thus, the nin5 deletion would be expected to remove the ribosome binding site located 7500 bases from the right end, and not to remove the ribosome-binding site located 8190 bases from the right end. The extent of the nin5 deletion is shown in Fig. 3 assuming nin5 is located between 5200 and 8000 bp from the right end of ~ DNA. A total of seven ribosome-binding sites would be removed by this deletion. If a gene is defined by the ribosome-binding site 8190 bases from the right end of ~ it would probably also be inactivated. Two ribosome binding sites
between the right end of nin5 and the gene presumed to be Q would not be removed by the nin5 deletion. This assignment is consistent with the locations of ribosome-binding sites on ~nin5dk6. The ribosomebinding sites on the two DNA molecules can be aligned by assuming that the complex of ribosome binding sites observed between 9 1 0 0 - 1 0 0 0 0 bases from the right end of kpk3 corresponds to the complex of ribosome-binding sites located 6300-7200 bases from the right end of knin5dk6. The difference between these values, 2800 bases, corresponds to the length of the nin5 deletion. If the bound ribosome on knin5dk6 located between 5000 and 5500 bases from the right end of DNA are assumed to define a single ribosome-binding site lying to the left of nin5, this ribosome-binding site would probably correspond to the ribosomebinding site located on kpk3 8190 bases from the right end of ~ DNA. The profile of the bound ribosomes at this position however suggest that on knin5dk6 there may be two very close ribosomebinding sites. These could be two ribosome-binding sites brought close together by the nin5 deletion. If so, the other ribosome-binding site is likely to be the one located on kpk3 5060 bases from the right end of k DNA. Other possible locations for the nin5 deletion that are consistent with the data for the locations of ribosome-binding sites on kpk3 and knin5dk6 fit the measured location of nin5 less well. In summary, it appears that there are about 20 ribosome-binding sites located to the right of genes O and P. A ribosome-binding site which may correspond to ren lies about 200 bases left of the left end point of nin5. The nin5 deletion would probably remove coding sequences for a gene associated with this ribosome-binding site, as was found for ten (Toothman and Herskowitz, 1980). Seven ribosomebinding sites are removed by the nin5 deletion. Two ribosome-binding sites may exist between the right end point of nin5 and gene Q. Genes in this region may be non-essential since the PQA4 deletion extends nearly to gene Q (Sternberg et al., 1979). A ribosomebinding site has been tentatively assigned to gene Q and another to the 6S region of ~,. Ribosome-binding sites likely to correspond to gene S and Rz have been located near transposons which inactivate these genes. A ribosome-binding site which may correspond to gene R lies between the sites assigned to genes S and Rz. Beyond gene Rz are four or five ribosome-binding
175 sites which may indicate the presence of up to five new ~ genes transcribed as part of the late mRNA.
ACKNOWLEDGEMENTS We thank Ryland Young for his phage strains and for helpful advice during the course of this work. We thank
Charles
Roessner
and
Leslie Merritt
for
assistance with ribosome and tRNA purification and for assistance with the electron microscopy. This work was supported by the National Institute of Health (grant R01 GM 24432).
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Hopkins, N.: Bypassing a positive regulator: isolation of a x mutant that does not require N product to grow. Virology 40 (1970) 223-229. Ihler, G. and Nakada, D.: Selective binding of ribosomes to initiation sites on single-stranded DNA from bacterial viruses. Nature 228 (1970) 239-242. Ihler, G. Calame, K. and Nakada, D.: Electron microscopic mapping of ribosome-binding sites on single-stranded DNA. Methods Enzymol. 59 (1979) 837-851. Lewin, B. Gene Expression, Vol. 3, Plasmids and Phages. Wiley, New York, 1977, pp. 274-535. Mark, K.-K.: Effect of the N-bypass mutations nin and byp on the rightward transcription in coliphage h. Mol. Gen. Genet. 124 (1973) 291-304. Pagano, T.S. and Hutchinson, C.A.: Small, circular, viral DNA: Preparation and analysis of SV40 and ~X174 DNA. Methods Virol. 5 (1971) 79-123. Scherer, G.: Nucleotide sequence of the O gene and of the origin of replication in bacteriophage lambda DNA. Nucl. Acids Res. 5 (1978) 3141-3156. Schwarz, E., Sherer, G., Hobom, G. and KOssel, H.: Nudeotide sequence of cro, cII, and part of the O gene in phage h DNA. Nature, 272 (1978) 410-414. Shine, J. and Dalgarno, L.: The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71 (1974) 1342-1346. Shizuya, H., Brown, D. and Campbell, A.: DNA polymerase I-independent mutants of coliphage lambda. J. Virol. 13 (1974) 947-952. Sklar, J., Yot, P. and Weissman, S.M. : Determination of genes, restriction sites, and DNA sequences surrounding the 6S RNA template of bacteriophage lambda. Proc. Natl. Acad. Sci. USA 72 (1975) 1817-1821. Sternberg, N. and Enqulst, L.: Analysis of coliphage lambda mutations that affect Q gene activity: puq, byp, and nin5. J. Virol. 30 (1979) 1-13. Sternberg, N., Hamilton, D., Enquist, L. and Weisberg, R.: A simple technique for the isolation of deletion mutants of phage lambda. Gene 8 (1979) 35 -51. Szybalski, E.H., Honigman, A., Rosenvold, E.C., Fiandt, M. and Szybalski, W.: A deletion in the P-Q (ninR) region of phage hb2imm21 conferring partial gene N21 independence. Gene 2 (1977) 295-297. Szybalski, E.H. and Szybalski, W. A comprehensive molecular map of bacteriophage h, Gene 7 (1979) 217- 270. Toothman, P. and Herskowitz, I. Rex-dependent exclusion of lambdoid phages. Virology (1980) (in the press). Young, R., Way, J., Way, S., Yin, J. and Syvanen, M.: Transposition mutagenesis of bacteriophage lambda: a new gene affecting cell lysis. J. Mol. Biol. 132 (1979) 307322. Westmoreland, B.C., Szybalski, W. and Ris, H., Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy, Science, 163 (1969) 1343-1348. Communicated by W. Szybalski.