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Electron microscopic mapping of RNA polymerase binding to coliphage lambda DNA
J. Mol. Bid.
(1078)
123, 485-498
Electron Microscopic Mapping of RNA Polymerase Binding to Coliphage Lambda DNA H. J. VOLLENWEIDER
f/niversity
AND W. SZYBALSXI
NcArdlc Laboratory for Cancer Research of W,iseonsin, Madison., Wise. 53’iO6, U.S.A.
(Received 6 January
1978, and in revisrd
,fom
21 March 1978)
Twenty Es&e&&a coti RNA polyrnerase molen~&s billci specifically Tao liriear, double-stranded colipbage h DNA at 30: 1 polymcrase-to-X DNA molar ratio, under the conditions used. ‘I’be binding sites ww identified by electron microtechrGcpe which measures binding scopy employing a gl~lutaraldellyde/I3ACt with 8476 specificity. Binding sites could be assigned to all well identified h promoters, including pI, p,. P,.,,,, 11)~. p, and pa, :rlt.hough only o,,e bound polgmerase could be found in tllc- ~+~-pa region, probably reflecting the low affinity Several binding sites seem to corresof RNA polymerase for t)be p rm promoter. polld to minor in vioo-active transcriptional startpoints (e.g., of lit or mis RNA), to potential promoter sites (e.g., hfip), or t,o the startpoints observed only during 2 and the 96 to 99.5% A its vitro enzymatic RNA synthesis (e.g., the 112 re+n region). Moreover, a. few binding sites are in t.llta regions t,ba.t bear no known startpoints but conbain known transcriptional tcarrninators. (‘orrelation between tile efficiency of initiation of RNA synthesis urltl the freqrlency of RNA polynrerase binding is good only for some promoters. all of the RNA polymerasc binding sites lie within the A /- T-rich regions, as determined by partial denaturat~on mapping. However, quantitative correlatiorl brtween frequencies of polyrnerase binding and localized DNA melting is far froltl pcrfec*t.
1. Introduction DNA of bacteriophage X is one of the most extensively studied molecules from the genetic and physical points of view. The physical maps, as derived from electron micrographic heteroduplex mapping, partial denaturation analysis and localization of the restriction endonuclease cleavage sites, were correlated with the transcriptional maps obtained from experiments both in vivo and in vitro (see Szybalski, 1976,1977). The positions of RNA polymerase binding sites on X DNA have been determined indirectly by a variety of techniques, e.g., binding to DNA fragments (Jones et al,, 1977), protection of ;\ DNA from endonucleolytic cleavage (Allet & Solem, 1974), including sequencing (Maniatis et al., 1975; Pirrott*a, 1976: Ptashne et al., 1976), and visualization or transcriptional mapping of very short transcripts (Botchan, 1976 : Blattner & Dahlberg, 1972; Blatt,ner et al., 1972,1974). Direct visualization of RNA polymerase binding by electron microscopy was performed by Hirsh & Schleif (1976) but only for a short DNA fragment carrying the pR and prm promoters. The
t Ahbreviationti
used: EUC, benzyldimothylalkyl-C,,,(‘,.,-ammonium
chloride.
485 0022~2836/78/1233-8698
$02.00/O
c) 1978 Academic
l’ress Inc. (London)
Ltd.
486
H. J. VOLLENWEIDER
AND
W.
SZYBALSKI
Since techniques were developed to determine with considerable precision the sites of RNA polymerase binding to double-stranded DNA, and no detailed data were available on the location of all the strong RNA polymerase binding sites on I\ DNA and on the affinity of the enzyme for the individual sites, we undertook to map carefully all such sites on the h DNA molecule and to determine whether such binding sites correspond to the positions of known promoters or startpoints of transcription. We found that this is generally true, but that there are also several RNA polymerase binding sites which might either have no biological role or correspond to other transcriptional signals, e.g., terminators. Altogether, 29 sites of stable RNA polymerase binding were localized on the X genome. The relationship of these binding sites to the known transcriptional control regions (Dahlberg & Blattner, 1973) and to the partial denaturation map of h DNA (Inman & Schnos, 1979 and personal communication) was quantitatively evaluated.
2. Materials and Methods (a) Reagents
and enzymes
BACt was a gift from Bayer, Leverkusen, Germany. Glutaraldehyde sealed under nitrogen was obtained from Polysciences, Warrington, RI. All other reagents were analytical grade chemicals. Polystyrene latex spheres (0.481 pm & 0.33%) were purchased from Fullam, Schenectady, N.Y. Escherichia coli K-12 RNA polymerase holoenzyme (nucleoside triphosphate :RNA nucleotydyl transferase, EC 2.7.7.6) was a homogeneous preparation containing stoichiometric amounts of sigma subunit, and was kindly given by Dr R. R. Burgess. Glassware was autoclaved prior to use. Sterile plasticware was purchased from Falcon Plastics, Oshard, Cal. Buffer solutions were passed through Millipore filters (250 nm pore size) and autoclaved. (b) Phage propagation,
purification
and DNA
preparations
The techniques employed for preparing Xc185787 DNA were essentially those outlined by Blattner et al. (1974) and Bavre et al. (1971). The DNA (O-8 mg/ml) was stored in O-01 M-Nacl, 0.01 M-Tris.HCl (pH 7.9) at 4°C. Homogeneity of the phage was examined by equilibrium density gradient centrifugation in an analyt,ical ultracentrifuge Spinco model E. (c) Binding
of RNA
polymeraxe
to DNA
DNA (25 ,ug) and RNA polymerase (30 : 1 polymerase-to-h DNA molar ratio) were incubated at 37°C for 20 min in 100 ~1 of a buffer containing 0.03 M-triethanolamine*HCl (pH 7.9) (TEA), 0.05 ~-Kc1 and 0.01 M-MgCl,. The complex was then treated by a a-step of the complex (10 min, 37°C) by adding procedure (Vollenweider et al., 1976) : (1) fixation 0.2 vol. of fixation buffer (0.5% glutaraldehyde in the above TEA/KCl/MgCl, buffer at pH 7.9; Koller et al., 1974; Portmann et al., 1974) and (2) opening the cohesive ends of /\ DNA by adding 1 vol. of dissociation buffer (7 y. formaldehyde and 0.1 y. glutaraldehyde in TEA) and raising the temperature to 57°C for 20 min. The next step was the purification of the RNA polymerase-DNA complex on a Sepharose 4B column, The complex was oluted from the column with an elution buffer (0.1% glutaraldehyde in TEA). The absorbance of the eluting material was measured at 260 nm. The DNA-containing fraction was used for electron microscopy. (d) Specimen
preparation
for electron microscopy
The procedure followed was as described by Vollenweider et al. (1975) with the stock solution of BAC diluted in elution buffer. The DNA or RNA polymerase-DNA complex was adsorbed to carbon film-coated copper grids placed on a 50-~1 droplet containing t See footnote
to p. 485.
RNA
POLYMERASE
BINDING
TO (IOLIPHAGE
h DNA
487
0.2 rg DNA/ml, 2 x 10e4% BAC, and 0.1% glutaraldehyde in TEA. The specimens were then washed by floating on redistilled water for 10 min. After dehydration of the specimens in 90% methanol or 90% ethanol or by freeze-drying (Vollenweider e$ al., 1978) the grids were rotary shadowed with uranium oxide (Westmoreland et al., 1969) and then sprayed on the same side with latex spheres in ethanol for magnification calibration. (e) Electron microscopy Electron micrographs (Kodak electron image plates) were taken with an Hitachi Hu- 11B at 75 kV, at 10,000 x magnification. Orientation of the grids was always the same and the focusing was on t,he DNA or uranium oxide granules bnt never on tile latex spheres. (f) Quan,titative
analysis
of the micrographs
Electron micrographs were projected with 13 x enlargement and the contour lengths of tile DNA molecules, the RNA polymerase binding sites and the circumferences of the latex spheres were measured with a vertically mounted electronic graphic calculator with it Numonics Corp., Lansdale, Pa). The counter-balanced arm (Mark 3, 264-3605-136; measuring accuracy of this device was about t 0.5:,,, determined hp measuring the same object several times.
3. Results (a) I’isualization
of RNA
polymeruse
h&ding
E. coli RNA polymerase exhibits a general affinity for h DNA. but under the conditions employed (see Materials and Methods) only a limited number of polymerasr molecules is retained on A DNA (Fig. 1). The important features of our technique as applied to X DNA include: (1) use of glutaraldehyde at low concentrat)ion (O*lO,/,) to link preferentially to DNA the specifically bound RNA polymerase molecules, while inactivating those in solution ; (2) the dissociation of h DNA concatenates by formaldehyde treatment (at high concentration of 359;), in order to facilitate thtb gel filtration and to free the cohesive h termini, which served as primary reference points in the electron microscopic measurements; and (3) removal of the unbound RNA polymerase molecules by gel filtration on Sepharose 4B. It was necessary to apply the two-step aldehyde treatment (0.1% followed by 350/;,), as introduced by Vollenweider et al. (1976), since immediate exposure of the RNA polymerase-DNA complexes to the high concentration of formaldehyde results in the loss of most RNA polymerase bound, as also confirmed by Cherny et al. (1977). For DNAs which do not have cohesive ends, step (2) could be omitted (Koller et al., 1978). The globular molecules associated with A DNA in Figure 1 must be RNA polymerase, since such complexes were observed only when E. coli RNA polymerase was added. Also, the glutaraldehyde-pretreated E. coli RNA polymerase (0.104 glutaraldehyde, 10 min, 37°C) does not associate with DNA, indicating that only the polymerase already bound to DNA can be fixed by such treatment. (b) Mapping (i) Orientation
of RNA
polymerase
hindi,rg sites
and general mapping
Under the present conditions and with a polymerase-to-DNA molar ratio of about 30, up to 20 enzyme molecules are associated with each h DNA molecule, bound predominantly to one arm of the genome. Since most of the h promoters are located on the right arm, we assumed that, this is the arm which selectively binds RNA polymerase. and correspondingly we oriented the h molecules on the histogram shown in
488
H.
J.
VOLLENWEIDER
AND
W.
FIG. 1. Electron microgmphs of the E-. coZ% RNA polymerasc hyde/BAC technique, as described in Materials and Methods. preparations.
SZYBALSKI
bound to X 1)NA. The glutaraldr~. wax cmploy~tl in making thew
(a) 100 -
60-
60
70
80
90
IO
Per cent X length (“/.A mts) molrcule~ in relat,ion to the A genetic map. FIO. 2. Distribution of bound E. coli RNA polymerase Histograms (a) and (b) represent the left and right arms, respectively, of t,he entire h DNA molecule; (c) shows the refined mapping of the immunity and adjacent regions. The preparation of t,he RNA polymerass NDNA complexes is described in Materials and Methods. Employing the circummolecules were selected. The ference of latex spheres as a reference, only the full-lengt,h h DNA positions of the binding sites were mapped as the percentage of t,hr total contour length of each DNA molecule (in y0 h units). Polymerases bound to the DNA t,rrmini were not recorded. Each bar corresponds to 0*5% A (240 baw-pairs assuming the h penome is 48,000 base-pairs; Vollenweider et al., 1978), with the exception of the broken lines in (b) which correspond to 0.25% X. In histograms (a) and (b), 327 DNA molecules were analyzed and the ponit.ions of a total of 3448 bound RNA polymerases were measured in relation to the right A DNA terminus. In histogram (c), molecules in 83 DNA molecules were selected for analysis, each containing 2 or more polymerase t~he 77.5 t,o 81 o/o A region; t,he rightmost polymrrase in this region tiewed as the reference point (SW Results and Fig. 3). These 83 DNA molecules carried a total of 1075 polymerase molecules, with 366 of them in the mapped segment. The relative pwitions of histograms (b) and (c) reflect t,ho alignment of the statistical means of t,he 91.to-93:/; h ~wak in (h) and the corresponding peak in (c). On the genetic map, t,he approximate posit,ions and orirntations of the known promoters are respectively (see Hlattner et nl., 1974; Szybalski, 1976; indicated by the letter p and an arrow, Pilacinski et al., 1977). The symbols lit (si startpoint and t, terminator) and mis indicate short transcripts (Hayes & Szybalski, 1973; Carlson et CL, 1978; vitrd by Szybalski, 1976) and hip a site where new promoters can be created by mutations (Salstrom ut rtl., 1978). The leftward arrows in the 44.5 t,o 57.6O/, h region and the rlght,ward arrows in t,he 50 to 650/; h region correspond to the in vitro transcriptional starts init,iat,ed with pppA or pppG (Rosrnvold, Blattner & Hzybalski, unpublished data). The approximate positions of known terminat,ors are indicat.od by thr symbols t (see Szybalski, 1976; Pilacinski et rrl., 1977; Salstrom K- Sxybalski, 1978).
H.
490
J. VOLLENWEIDER
AND
W.
SZYBALSKI
Figure 2. In this histogram 327 intact X genomes associated with 3448 RNA poly merase molecules were analyzed. Figure 2(a) and (b) represents RNA polymerase binding to the left and right halves, respectively, of the entire X DNA molecules. The positions of the RNA polymerase binding sites were measured in respect to the right X terminus. The peaks correspond to the regions of most frequent polymerase binding, mainly in the center of the h genome and in several sites on the right arm. It is clear that under the present conditions RNA polymerase binds selectively to specific regions of the X genome. (ii) Background
of non-speci$c
binding
Very few polymerases bind to the left arm of X between 0 and 40% X, and since no known h promoters are located in this DNA region, we assume that these randomly bound polymerases represent the experimental background. If this background level is uniform throughout the entire h genome, we could calculate that under our conditions 16% of RNA polymerase binding is non-specific. (iii) Refined mapping
in the immunity
and adjacent regions
Since many important regulatory sites are located in the neighborhood of the h immunity region within the 70 to 90% X segment and several peaks of RNA polymerase binding are observed in this region (Fig. 2), a reference point located nearer to this region than the right X terminus was employed to shorten the measured distances and hence reduce the error. The position of the rightmost of two polymerase molecules (see Fig. 3) that bind to the segment between 775 and 81% X (Fig. 2(b)) was chosen as such a reference point, as indicated by the bar in the 79.5 to 80.0% interval (Fig. 2(c)). This procedure permits the resolution of several peaks in Figure 2(c), which appear to correspond to known X control elements in this region (see Discussion) and which were not discernible in Figure 2(b). (c) Analysis
of individual
RNA
polymerase binding regions
Examination of the histograms shown in Figure 2 reveals that RNA polymerase molecules bind to defined regions of the X genome represented both by the narrow and broad peaks of different heights. It is not clear, however, whether the broadness of a given peak indicates variability in the position of the binding site, inaccuracy of the measurements, or the presence of multiple and closely spaced binding sites. We therefore analyzed how many RNA polymerase molecules could simultaneously bind to each particular DNA molecule within the regions corresponding to the peaks. The results of this analysis, which comprised all the 327 DNA molecules represented in Figure 2 (with the 16% background of the non-specific RNA polymerase binding subtracted, as represented by the shaded areas), are shown in Figure 3. Some regions, as e.g., the 91 to 93% A segment, bind only one polymerase, whereas others, as e.g., the 44.5 to 58.5% X region, which comprises the A + T-rich b2 region, bind up to eight RNA polymerase molecules (Fig. 3, column 3). Column 6 represents the results of more refined mapping (Fig. Z(c)), which permitted resolution of the 77.5 to 8l*Oo/o X segment into two smaller regions, each binding only one polymerase. Similarly, the 69.5 to 77*Oo/o h segment was resolved into three separate regions. These results indicate that about half of the regions bind only one RNA polymerase for which a position can be statistically assigned (Table 1). The remaining regions
RN.4
POLYMERASE
BINDING
Number of RNA polymemses
TO
wlthin
Dato used in Fig. 2(a).(b)
COLIPHAGE
mdwduol
X DNA
491
bmdmg regkons
Doto used in Fig. 2(c)
I I
Uo. of polymeroses
No. of DNA molecules
region (%A) 1
60.0-69.5
1p
:
No. of polymeroses
No. of DNA molecules
I
I
P
5p
‘90
‘5,OI regbm (%A)
0
50
IOC
I
77*0-79,3 79.0
96.0-99.5
1;
1 I,
FIO. 3. Number of RNA polymerases within individual binding regions of h DNA. The binding regions (column 1) correspond to thr polymerase peaks on t,he X map in histograms (a) and (b) of Fig. 2. Each of the 327 A DNA molecules was analyzed individually, and the frequency of DNA molecules per region (column 2) is molecules simultaneously carrying 1, 2, 3 or more polymerase represent,ed in the form of separate histograms in column 3. The shaded portion of each histogram corresponds t>o thv experimental background of 16%, i.e. 5.5 polymerases per 1 o/0 A. The results in columns 4 to 6 arc presented in an analogous way using the data from Fig. 2(c), with the experimental background corresponding to 1.7 polymrrases per 19 h.
seem to correspond t’o clusters of two or more binding by th(x present, electron microscopic analysis. (d) Total number of RNA polyrnerase
sites that could not be resolved
binding
site.8 on h DNA
Twcsnty RNA polymerase molecules bind specifically to h DNA at a 30: 1 polymerase-to-h DNA molar ratio under the conditions used. This number is derived by adding the highest number of polymerase molecules bound in each region, after discounting the non-specifically bound polymerases indicated by shaded areas in column 3 of Figure 3. The number 20 remains unchanged when the data in column 6 are used.
492
H.
J.
VOLLENWEIDEH.
AND TABLE
W.
SZYBALSKl
1
RNA polymerase binding sites, promoters and some other regulatory &es on A DNA
Boundaries of the X DNA region ( y0 h)
No. of RN.4 polymcrase molecules bound wit,hin a region
44.5-58.5
x
58.5-62.0
2
68.0-69.5 70.3-71.8 72.8-73.8 73.8-75.8
1 1 2
77.8-79.3
1
794% 91.0-93.0 96.0-99.5
1 1 2
C’alculatrd position of the RNA pol,ymwasc? binding sitrt (O/” ,I)
Position and designation of t,hr h promoters, st,art,points and other transcriptioncontrolling elements (“/;, h)$ b2 rogion : two major 1 pppA startpoints two major I pppG startpoints one major T pppG startpoint 60.4; p, one T startpoint 68.6; hip, t,, il.1 ; f,, 73.5; ,‘L 75.7; lit, wis startpoint 74.3; ti 78.4; prm 78.5; *Ia 79.9; 1,” !12.3 ; p’*$ startpoirlt(s) OLL supcrhelical DN.4
h
The boundaries (column 1) were chosen to fit the valleys bet,wcrn the peaks. The number of RNA polymerase molecules (column 2) corresponds to tho highest nnmbcr in columns 2 and 5 of Fig. 3. t Statistical mean for all RNA polymerascs located in a given wgion. C’onfidenco interval calculated at 0.99 confidence level. i See legend to Fig. 2 and Discussion (section (c)). f Used as reference point in Fig. 2(c).
4. Discussion (a) XpeciJicity
of the glutaraldehyde/BAC
technique
When employing the glutaraldehyde/BAC technique, the specificity of RNA polymerase binding to X DNA was high, since only 160/, of non-specific binding was observed. In contrast, about 55O’,,0 of non-specific binding of RNA polymerase was seen in the absence of any fixatives or competing agents (Hirsh & Schleif, 1976). After our study was completed, a very similar result was also reported in the Appendix to the study of Williams & Chamberlin (1977). In their case use of 0.1 y0 of glutaraldehyde decreased the unspecific RNA polymerase binding by 35q& as compared with the glutaraldehyde-free controls. Our figure of 16% was derived by scoring the number of RNA polymerases on the left arm of X DNA, which does not contain any known transcriptional control elements. We recognize that the G + C content is higher in this arm than in other regions of X DNA ( see review of Davidson 8z Szybalski, 1971), but the base composition per se should not affect the RNA polymerase binding. For example, the residual and non-specific binding i?j, vitro of E. coli RlVA polymerase to the right arm (50-to-80%) region of the T7 genome is random (Port’mann et al., 1974; Koller et aE., 1978), although this DNA region contains distinct segments with high A + T content, as shown by partial denaturation (G6mez & Lang, 1972).
RN.4
POLYMEHARE
HIh’JlrNG
TO
COLJPHAGE
X IlRTA
4!K3
The reasons for the enhanced specificity may be manifold, but we shall consider two likely alternatives based on thth following schematic prewntations:
ONA t RP
I_ -2
ONA*RP
3 I +o
ONA
+ RP 4
4 1 +a
5
DNAmRIP P
-
6
“““‘97 P
If glutaraldehyde (g) reacts with free RNA polymerase (RP) reaction 3 results in inactivation of the enzyme (SW Results), which \\hen so modified cannot dissociate, into subunits (H. Bujard, personal communication). Reaction 4 is the first-step reaction between glutaraldehyde and RNA polymerase molecules bound to DNA. In case of non-specifically bound enzyme this fir&-step reaction may result in displacement of the polymeraac (reaction 5) since binding was weak (see Chamberlin. 1974). The second possibilit)y is that t’he enzyme would dissociate anyway (reaction 2 or 5) before t’he second-step fixation reaction 6 occurs, especially since the dwelling time of the non-specifically bound enzyme on DNA is short (see Chamberlin, 1974) as compared with the time required for react’ions 4 and 6 (Richards & Knowles, 1968 : Monsan et al., 1975). In the case of the specifically bound enzyme, the fixation (reaction 6) will br favored, sinw the dwelling time is much longer a,nd binding much stronger (see Chamberlin. 1974). To increase the specificity of RI\‘A polymerase binding for electron microscopic mapping, ot,her invest’igators used either poly( I) or heparin. Poly(1) appears less suitable since it permits about’ 25();, non-specific binding (data calculated from Fig. 5 of Hirsh & Schleif, 1976), whereas we observed only IS:;, for glutaraldehyde. It was not possible to estima,te the efft~ctiveness of hrparin from the data of Giacomoni ef nl. (1977). The frequency of RNA polymerase binding (nun] her of RNA polymerases visualized at each specific binding site, as shown in Figs 2 and 3) varies from one specific binding site to anot’her. Factors responsible for this variation (see scheme) may include (i) number of specifically bound RKA polymera,ses at the time of glutaraldehyde addition. (ii) the rates of dissociation (rewct)ion 2) and displacement’ 1)~ glutaraldehyde (r(‘action 5). and (iii) the rate of fixation (reactions 4 and 6). The rate of association (reaction 1) will become negligible soon after glutaraldehydc addit’ion since free RNA polymoraw will be inactivated b,v this fixative (reaction 3). Although we are not certain which of these factors an’ crucial in determining the frequency of RNA polymrrase binding to the h DNA, as visualized by the glutaraldehyde/BAC technique, thaw arc’ probably in common with t’hc fact#ors wsponsible for the retention efficiency of /\ DNA fragmcnt,s on nitrocellulose filtws in the presence of RNA polymerase and heparin (Jones ef ~2.. 19i7), becauw of the remarkable correlation between the rwults obtained by thew two methods (Fig. 4(b) and (c)). (I)) Comparison
with other methods of mapping
KflA
polymeruse
binding
sites
Early studies on RNA polymerase binding to h DNA led to the estimation of the total number of bound enzyme molecules, but not t’o their localization. For example.
H.
J. VOLLENWEIDER
AND
W.
SZYBALSKI
a)
b)
j-
:)
P
fmdll
I,
A&III
II
20
30
P
P
F
1
hdIIl IO
40
50
Per cent X length
60 (%A
70
00
90
I
mts)
FIG. 4. Comparison of the partial deneturation map of h UNA with the RNA polymerase binding map. (a) Partial alkali-denaturetion map kindly provided by Dr It. B. Inman and representing a refined version of the previously published map (Inman & Schniia, 1970) where t,he average deneturation per h molecule is 12.3%. (b) Map of E. co& RNA polymerese binding &es derived from Fig. 2 (a) and (b) with the experimental background subtracted. (c) Map of the (i) Hind11 and Hind111 cleavage sites represented by the downward or upward pointing arrow, respectively (Robinson & Lendy, 1977a,b); (ii) Hind11 and III fragments that bind E. coli RNA polymerese and are efficiently retained on filters at an 11: 1 polymerase-t,o-h molar ratio (black rectangles), inefficiently retained (shaded rectangles), or retained only at a 47: 1 molar ratio (open rectangles), when X DNA and RNA polymerese were incubated for 30 min at 37°C and then treated with an excess of heparin for 10 min at 37°C (Jones et nl., 1977); and (iii) the cleavage sites protect)ed by the bound RNA polymerase as indicated by symbol P (Jones et crl., 1977). Since XpZnc5 was used in these experiments, the region within brackets does not correspond to maps (a) and (b).
Nakano $ Sakaguchi (1969) reported that up to 18 polymerase molecules could bind to h DNA, in good agreement with the present results. Fragmentation of DNA by the restriction nucleases permitted the assignment, of RNA polymerase binding sites to those X fragments retained on nitrocellulose filters. The results of such a study by Jones et al. (1977) are shown in Figure 4(c). It can be seen that there are at least nine fragments that bind polymerase, four of them with the highest efficiency and two with the intermediate one. The location of those fragments on the h map and the efficiency of their retention (measured as the required polymerase-to-DNA ratio; Fig. 4(c)) a g ree well with our data as represented by the positions and heights of the peaks (Fig. 4(b)). It might be interesting to note that some fragments were retained on the nitrocellulose filters only at RNA polymerase/h DNA ratios as high as 47 : 1 (open rectangles in Fig. 4(c)), whereas we could visualize same RNA polymerase binding at probably the same sites already at a ratio of 30 : 1.
KS.1 POLYMEHAHE
BINT)ING
TO C'OLlf'H;\GE
A DSA
Other data, based on the protection of restriction sites and initiation synthesis, will be considered in the following sections. (c) Comparison, of the positions of the RiVA polymerasp with those of the known promofers
binding
495 of RNA
sites
The binding of RNA polymerase to X DNA. as discussed up to this point, did not imply any biological funct~ion for this interaction. However, it is known that RNA polymrrase binding is one of the steps in the initiation of t’ranscription, and t,he availability of detailed maps of t,he in viva and i,r vitro initiation sites in h permits the determinat,ion of which of the binding sites represent t,he active promoters. The positions and orientation of the known promoters are indicated by the horizontal arrows for the left (Fig. 2(a)) and right arms (Fig. 2(b) and (c)) of h DNA. Witlr few exceptions the correlation between the peaks indicating the binding regions and the positions of the promoters is very good. On the left arm there are no peaks of specific ROTA polymerase binding and there are no know,n promoters (Fig. 2(a,)). The first two peaks between 44.5 and 585°{I /\ correspond to several leftward and right ward startpoints observed during 20 seconds of ire vitro transcription in t,he A + Triclr hd region of h, as determined by Rossnvold: Hlattner & Szybalski (unpublished data). Their lower estimat’e for t,he number of strong startpoints is five. whereas wc observe eight binding sites in this region (Fig. 3). Proceeding rightward. a small peak of RN,4 polymerase binding could be observed to two binding sites (Fig. 3), in the 58.5 to 62.0% A region (Fig. 2(b)). corresponding which could represent the ~pr promoter located at 60.4’$; /\ (Honigman & Hu? 1978: Pilacinski et aZ., 1977) and a righhward start observed in rrifro by Rosenvold. Blat’tner & Sz,vl)alski (unpublished data). The major pL and pR promoters correspond to the single binding sites at 73.3 and (Fig. 2(b) and (c), and Table 1). The p0 promoter for 0023RNA 78.4”,‘, /1, rrspect,ively corresponds to the single binding sit’e at 79.8 9,;)h. The agreement between our measurements and the sequencing data is excellent since we measured the distance between are 653 base~1~and p,, as 650&35 base-pairs. whereas t’he corresponding startpoints pairs apart according to sequencing data of Schwarz et al. (1978). We saw only one polymerase binding site in the ~I,.~-P~ region and the simplest’ interpretabion is that under our conditions only very few or no RNA polymerases bind to the p,., promoter. When st,udying the protection of the prm and pR sites from nucleolytic digestion under comparable conditions (Meyer et al.. 1975), B. Meyer (personal communication) observed RNA polymerasc binding to the p,, promoter only when t,he ratio of polgmerase to the prrn promoter was ovtr 20. whereas in om case it \vas less than 2 (30 polymerases per 20 binding sites). We observed two polymerasc binding sit)es located to t’hr left of yL, one at 68,6 and another at 71.00,:, h. The tirst one corresponds either t’o t,he position of the t,, leftward terminator or to the site where point’ mutations could easily generate a new left(ward promoter, designated hip (Salstrom & Szybalski, 1978: Salstrom et al., 1978). The second. at 71.0% X corresponds to the position of the t,, leftward terminator site (Salstrom & Szybalski, 1978). We do not know ðer t’he present’ly observed RKA polymerase binding near or at the terminators could have some role in the termination function. It is unlikely that strong polymerase binding should be a general feature of the terminat,ors. since we did not observe any strong binding at the site corresponding
H.
496
J.
VOLLENWEIDER
AND
W.
SZYBALSKI
to the third strong leftward terminator, t,,, located at 64.4% h (Salstrom t Szybalski, 1978). We observed two binding sites within the middle of the h immunity region at 73% to 75.8% h, but their relationship to the startpoint (sr) or terminator (ti) of the in viwo produced lit transcript located between 74.3 and 75.6%) \ (Hayes & Szybalski, 1973) or to the in vitro-synthesized m,is transcript (Carlson et al., 1978 and cited by Szybalski, 1976) is unknown. The single binding site at 92.0&O-l “/o h (Table 1) shows excellent correspondence to the position of the startpoint controlled by the pk late promoter calculated to be at 92.3% X as based on the data of Sklar et al. (1975) and Robinson $ Landy (1977a,b). The remaining two binding sites in the 96.0 to 99*50/b h region do not correspond to any known X control sites that operate in vivo. However, $llet & Solem (1974) detected RNA polymerase binding in this region which protected one site from Hind11 endonucleolytic cleavage (see also Jones et al., 1977), and Botchan (1976) has been able to force initiation of in vitro transcription in this region by using supercoiled circular h DNA templates. The latter data do not permit one to distinguish between one or more transcriptional startpoints in this region. The data discussed in this section are summarized in Table 1. (d) The number of sites, the frequency of RNA polymerase bind&g the eficiency of in vitro transcriptional starts
sites and
In several studies the initiation of transcription was used as a method for scoring the polymerase binding sites on h DNA. For example, Nakano & Sakaguchi (1969) observed 17 sites on which RNA polymerase initiates transcription and Willmund & Kneser (1973) estimated that there are six to 11 or more such sites. Furthermore. data listed in Table 1, which indicate eight known transcriptional starts on t,he Xl12 template (i.e. outside the 62 region). together with the results of Rosenvold, Blattner & Szybalski (unpublished data) showing live startpoint’s in the b2 region, add up to 13 polymerase initiation sites. Thus, the number of active initiation sites seems to be somewhat lower than the total number of binding sites observed in our study (Table 1). In Table 2 we compared the frequency of polymerase binding at the sites corresponding to the four known promoters, (I)~, pR, y0 and pa, with the efficiency of initiation of in vitro transcription at these promoters, as calculated by Dahlberg & Blattner TABLE 2 Frequency
of RNA polymerase bindkg and eficienc?/ of in vitro trarwription at Jive binding sites on X I)NA RNA
0/0 binding of RNA polymeranet T/, in vitro initiation of RNA synthesis by RNA polymerase$
polymcrase
binding
PI.
1’11
PO
21 22
2I 24
16 (i
Numbers in each line add to 1000/b. t Derived from data in Fig. 2. $ Based on data of Dahlberg & Blattnw
(1973)
for linear
sites and promoters 9G-99.5% PK 20 48
h DNA.
initiation
oj
X region ‘3
RNA
POLYMERASE
BINDlNG
TO
COLIPHAGE
h DNA
497
(1973). The correlation is very good for the p, and pR promoters, but there is a morel than twofold difference at the p, and & promoters. No initiation of transcription in the 96 to 99.50,/, h region was reported by Dahlberg & Blattner (1973), huh as already mentioned, Botchan (1976) was able to force in vityo transcription in this region on a supercoiled DNA template. The present results show that) supercoiling is not required for an ef?icient binding of RNS polymerase, but it is not clear whether it aids in the initiation of transcription or abolishes some closc~ly associated termination event. (e) Relationship
betwee?%the pol?ymerase hindirly
sites ad
A + T-rich
regions
There appears to be a remarkable correlation between the position of the RR’A polymerase binding sites and the A + T-rich regions, the latter determined by partial denaturation mapping (Fig. 4). Similar conclusions were recently reached both for h DNB (Jones et al., 1977; Botchan, 1976) and for coliphage Ml3 DNA (Dasgupta et ad.. 1977). Close inspection, however, reveals an imperfect quantitative correspondence for a few regions. For example, the ratio of peak heights in regions 91 to 93 and 96 to 99.5s/, h in Figure 4(a) is inverse to that in Figure 4(b). Some of the most prominent peaks of polymerasc binding and denaturation bubbles are not associated with any known in vivo acting promoters, alt’hough in vitro initiat,ion of transcription was observed in these regions (see regions 44.5 to 58.57; h and 96 t,o 99*5’:/, ,I). Furthermore, F. coli RNA polymerasc? does not bind to d + T-rich regions per se. as already discussed for the T7 phage genornc (Keller et al., 1978: G6mez & Lang, 1972). These data strongly suggest that high A--T contcant is an important but not sufficient criterion for specific and &Gent initiation of transcription.
We t,hank Mrs A. James for excellent technical assistancc~. 111’ R. B. Inman generol~sl> supplied us with the most current denaturation map of A DN,l (Fig. 4(a)) and Dr R. 1~. Burgess with the E. coli RNA polymerase. lsTe thank Drs R. R. Burgess and T. Koller for critically reading the manuscript and Dr E. Szybalski for editorial help. We also express our appreciation to Lois Wilson and
REFERENCES Allet, 13. 85 Solem, R. (1974). J. ~21ol. Biol. 85, 475-484. Blatt,rler, F. R. & Dahlberg, J. E. (1972). lVa,ture Nev* .Gol. 237, 227-232. Blattner, F. R., Dahlberg, J. E., Boettiger. J. K., Nantit, M. & Szpbalski. W. (1!)72). Nature New Biol. 237, 232.-236. Blattner, F. R., Fiandt, M., HRSS, K. R., Tn-osr. 1’. A. S- Szyba,lski, W. (1974). l’irolo~gy, 62, 458-471. Botchan, I’. (1976). J. &foZ. Bid. 105, 161-176. Bsvre, K., Lozeron, H. A. & Szybalski, W. (I 971). In &‘ethods in I~irology (Maramorosch, K. & Koprowski, H., eds), vol. 5, pp. 271-292, Academic Press, New York. Carlson. K., Krill, J. & Szybalski, W. (3978). illul. Gen. Gevet. in tlw press. Chambcrlin, M. J. (1974). Ann~u. Rev. Biochem. 43, 721-775. Cherny, D. I., Alrksandrov, A. A., Zarudnaya, M. I., Kosa~anov. ‘I’. N., Lazurkin, Y. S., Bmbealashvilli, R. S. & Savochkinn, L. I’. (1977). Eur. .I. Biochem. 79, 309-317. Dahlbera, .J. E. & Blattner, F. K. (1973). In I’imrs Resenrch, 2nd 1. c:. N.-U.C.L.A. Syrrrp. on Mol. Biol. (Fox, (:. F. & Robinson, TV. S., vtln). pp. 5X&543, Academic Press. New York.
498
H.
J.
VOLLENWEIDER
AND
W.
SZYBALSKI
C. E. Kt Mitra, S. (1977). .J. BioZ. Chem. 252, Dasgupta, S., Allison, D. P., Snyder, 5916-5923. Davidson, N. & Szybalski, W. (1971). In The Bacteriophage h (Hershey, A. D., ed), pp. 45-82, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Giacomoni, P. U., Delain, E. & LePecq, J. B. (1977). Eur. J. Biochem. 78, 205-213. G6mez, B. & Lang, D. (1972). J. Mol. Biol. 70, 239-251. Hayes, S. & Szybalski, W. (1973). Mol. Gen. Genet. 126, 275-290. Hirsh, J. & Schleif, R. (1976). J. &foZ. Biol. 108, 471-490. Honigman, A. & Hu, S.-L. (1978). IT’iroZogy, in the press. Inman, R. B. & SchnGs, M. (1970). .J. Mol. BioZ. 49, 93~.98. Jones, B. B., Chan, H., Rothstein, S., Wells, R. D. & Reznikoff, W. (1977). Proc. Nat. Acad. is., U.S.A. 74, 4914-4918. Koller, T., Sogo, J. M. & Bujard, H. (1974). Biopolymers, 13, 995-1009. Koller, T., Kiibler, O., Portmann, R. & Sogo, J. M. (1978). J. f!oZ. Biol. 120, 121-132. Maniatis, T., Ptashne, M., Backman, K., Kleid. D., Flashman, S., Jeffrey, A. & Maurer, R. (1975). Cell. 5, 109-113. Meyer, B. J., Kleid, D. G. & Ptashne, M. (1975).Proc. h’at. Acad.Xci., U.S.A. 72,4785- 4789. Monsan, P., Puzo, G. & Mazarguil, H. (1975). Biochimie, 57, 1281-1292. Nakano, E. & Sakaguchi, K. (1969). J. Biochem. 65, 147-150. Pilacinski, W., Mosharrafa, E., Edmundson, R., Zissler, .J., Fiandt, M. & Szybalski, W. (1977). Gene, 2, 61-74. Pirrotta, V. (1976). In Current Topics in Microbiology ad Immunology, vol. 74, pp. 21 -54, Springer Verlag, Berlin. Portmann, R., Sogo, J. M., Koller, T. & Zillig, W. (1974). FEBS Letters, 45, 64.-67. Ptashne, M., Backman, K., Humayun, M. Z., Jeffrey, A., Maurer, R., Meyer, B. & SRIIPI., R. T. (1976). Science, 194, 156-161. Richards, F. M. & Knowles, J. R. (1968). J. Mol. BioZ. 37, 231-233. Robinson, L. H. & Landy, A. (1977a). Gene, 2, l-31. Robinson, L. H. & Landy, A. (1977b). Gene, 2, 33-54. Salstrom, J. S. & Szybalski, W. (1978). T’irology, in the press. Salstrom, J. S., Fiandt, M. bi Szybalski, W. (1978). Mol. *en. Genet. in the press. Schwarz, E., Scherrr, G., Hohom, G. & Kiisscl, H. (1978). ilrature (London), 272, 410-414. Sklar, J., Yot, P. & Weisman, S. M. (1975). I’roc. Nat. Acad. Sci., U.S.A. 72, 1817-1821. Szybalski, W. (1976). In Handbook of Riochemhstry and Molecular Biology (Fasman, G. C., ed.), 3rd ed., N,zlcZeic Acids, vol. 2, pp. 677-685, CRC Press, Cleveland, Ohio. Szybalski, IV. (1977). In Regulatory Biology (Copeland, .J. C. & Marzluff, G. A., ods), pp. 3-45, Ohio Sta,te University Press, Columbus, Ohio. Vollenweider, H. J., Sogo, .J. M. & Keller. T. (1975). I’roc. Nat. Acarl. Sci.. T7.S.B. 72, 83-87. Vollenweider, H. J ., Koller, T., W eb er, H. 85 Weissmann, c’. (1976). ,J. ~%fol. Biol. 101, 367-377. Vollenweider, H. J., James, A. R: Szybalski, W. (1978). I’roc. Nat. AC&. &i., I.S.A. 75, 710-714. Westmoreland, B. C., Szybalski, W. & Ris, H. (1969). Science, 163, 1343-1348. Williams, R. C. $ Chamberlin, M. J. (1977). Proc. Nat. Acad. Sci., D.S..4. 74, 3740-3744. Willmund, R. & Knoser, H. (1973). ,?foZ. Gen. Genet. 126, 165-175. Note added in proof: Recently (W. Taylor and N. Burgess, unpublished 6.916 fragment (93.19 to loo?/,) situated to the right. of pa’ (Sklar shown to behave like other h promoter-containing fragments. It polymerase and is retained on a nitrocellulose filter along with other fragments. In addition, when ribonucleoside 5’-triphosphates are reaction, initiation can occur so that this fragment (like other fragments) is retained on the filter even after a 0.6 &r-KC1 wash.
results) the /\.%oRl et al., 1975) has been binds h’. coli RNA promoter-containing added to the binding promoter-containing
(1078)
123, 485-498
Electron Microscopic Mapping of RNA Polymerase Binding to Coliphage Lambda DNA H. J. VOLLENWEIDER
f/niversity
AND W. SZYBALSXI
NcArdlc Laboratory for Cancer Research of W,iseonsin, Madison., Wise. 53’iO6, U.S.A.
(Received 6 January
1978, and in revisrd
,fom
21 March 1978)
Twenty Es&e&&a coti RNA polyrnerase molen~&s billci specifically Tao liriear, double-stranded colipbage h DNA at 30: 1 polymcrase-to-X DNA molar ratio, under the conditions used. ‘I’be binding sites ww identified by electron microtechrGcpe which measures binding scopy employing a gl~lutaraldellyde/I3ACt with 8476 specificity. Binding sites could be assigned to all well identified h promoters, including pI, p,. P,.,,,, 11)~. p, and pa, :rlt.hough only o,,e bound polgmerase could be found in tllc- ~+~-pa region, probably reflecting the low affinity Several binding sites seem to corresof RNA polymerase for t)be p rm promoter. polld to minor in vioo-active transcriptional startpoints (e.g., of lit or mis RNA), to potential promoter sites (e.g., hfip), or t,o the startpoints observed only during 2 and the 96 to 99.5% A its vitro enzymatic RNA synthesis (e.g., the 112 re+n region). Moreover, a. few binding sites are in t.llta regions t,ba.t bear no known startpoints but conbain known transcriptional tcarrninators. (‘orrelation between tile efficiency of initiation of RNA synthesis urltl the freqrlency of RNA polynrerase binding is good only for some promoters. all of the RNA polymerasc binding sites lie within the A /- T-rich regions, as determined by partial denaturat~on mapping. However, quantitative correlatiorl brtween frequencies of polyrnerase binding and localized DNA melting is far froltl pcrfec*t.
1. Introduction DNA of bacteriophage X is one of the most extensively studied molecules from the genetic and physical points of view. The physical maps, as derived from electron micrographic heteroduplex mapping, partial denaturation analysis and localization of the restriction endonuclease cleavage sites, were correlated with the transcriptional maps obtained from experiments both in vivo and in vitro (see Szybalski, 1976,1977). The positions of RNA polymerase binding sites on X DNA have been determined indirectly by a variety of techniques, e.g., binding to DNA fragments (Jones et al,, 1977), protection of ;\ DNA from endonucleolytic cleavage (Allet & Solem, 1974), including sequencing (Maniatis et al., 1975; Pirrott*a, 1976: Ptashne et al., 1976), and visualization or transcriptional mapping of very short transcripts (Botchan, 1976 : Blattner & Dahlberg, 1972; Blatt,ner et al., 1972,1974). Direct visualization of RNA polymerase binding by electron microscopy was performed by Hirsh & Schleif (1976) but only for a short DNA fragment carrying the pR and prm promoters. The
t Ahbreviationti
used: EUC, benzyldimothylalkyl-C,,,(‘,.,-ammonium
chloride.
485 0022~2836/78/1233-8698
$02.00/O
c) 1978 Academic
l’ress Inc. (London)
Ltd.
486
H. J. VOLLENWEIDER
AND
W.
SZYBALSKI
Since techniques were developed to determine with considerable precision the sites of RNA polymerase binding to double-stranded DNA, and no detailed data were available on the location of all the strong RNA polymerase binding sites on I\ DNA and on the affinity of the enzyme for the individual sites, we undertook to map carefully all such sites on the h DNA molecule and to determine whether such binding sites correspond to the positions of known promoters or startpoints of transcription. We found that this is generally true, but that there are also several RNA polymerase binding sites which might either have no biological role or correspond to other transcriptional signals, e.g., terminators. Altogether, 29 sites of stable RNA polymerase binding were localized on the X genome. The relationship of these binding sites to the known transcriptional control regions (Dahlberg & Blattner, 1973) and to the partial denaturation map of h DNA (Inman & Schnos, 1979 and personal communication) was quantitatively evaluated.
2. Materials and Methods (a) Reagents
and enzymes
BACt was a gift from Bayer, Leverkusen, Germany. Glutaraldehyde sealed under nitrogen was obtained from Polysciences, Warrington, RI. All other reagents were analytical grade chemicals. Polystyrene latex spheres (0.481 pm & 0.33%) were purchased from Fullam, Schenectady, N.Y. Escherichia coli K-12 RNA polymerase holoenzyme (nucleoside triphosphate :RNA nucleotydyl transferase, EC 2.7.7.6) was a homogeneous preparation containing stoichiometric amounts of sigma subunit, and was kindly given by Dr R. R. Burgess. Glassware was autoclaved prior to use. Sterile plasticware was purchased from Falcon Plastics, Oshard, Cal. Buffer solutions were passed through Millipore filters (250 nm pore size) and autoclaved. (b) Phage propagation,
purification
and DNA
preparations
The techniques employed for preparing Xc185787 DNA were essentially those outlined by Blattner et al. (1974) and Bavre et al. (1971). The DNA (O-8 mg/ml) was stored in O-01 M-Nacl, 0.01 M-Tris.HCl (pH 7.9) at 4°C. Homogeneity of the phage was examined by equilibrium density gradient centrifugation in an analyt,ical ultracentrifuge Spinco model E. (c) Binding
of RNA
polymeraxe
to DNA
DNA (25 ,ug) and RNA polymerase (30 : 1 polymerase-to-h DNA molar ratio) were incubated at 37°C for 20 min in 100 ~1 of a buffer containing 0.03 M-triethanolamine*HCl (pH 7.9) (TEA), 0.05 ~-Kc1 and 0.01 M-MgCl,. The complex was then treated by a a-step of the complex (10 min, 37°C) by adding procedure (Vollenweider et al., 1976) : (1) fixation 0.2 vol. of fixation buffer (0.5% glutaraldehyde in the above TEA/KCl/MgCl, buffer at pH 7.9; Koller et al., 1974; Portmann et al., 1974) and (2) opening the cohesive ends of /\ DNA by adding 1 vol. of dissociation buffer (7 y. formaldehyde and 0.1 y. glutaraldehyde in TEA) and raising the temperature to 57°C for 20 min. The next step was the purification of the RNA polymerase-DNA complex on a Sepharose 4B column, The complex was oluted from the column with an elution buffer (0.1% glutaraldehyde in TEA). The absorbance of the eluting material was measured at 260 nm. The DNA-containing fraction was used for electron microscopy. (d) Specimen
preparation
for electron microscopy
The procedure followed was as described by Vollenweider et al. (1975) with the stock solution of BAC diluted in elution buffer. The DNA or RNA polymerase-DNA complex was adsorbed to carbon film-coated copper grids placed on a 50-~1 droplet containing t See footnote
to p. 485.
RNA
POLYMERASE
BINDING
TO (IOLIPHAGE
h DNA
487
0.2 rg DNA/ml, 2 x 10e4% BAC, and 0.1% glutaraldehyde in TEA. The specimens were then washed by floating on redistilled water for 10 min. After dehydration of the specimens in 90% methanol or 90% ethanol or by freeze-drying (Vollenweider e$ al., 1978) the grids were rotary shadowed with uranium oxide (Westmoreland et al., 1969) and then sprayed on the same side with latex spheres in ethanol for magnification calibration. (e) Electron microscopy Electron micrographs (Kodak electron image plates) were taken with an Hitachi Hu- 11B at 75 kV, at 10,000 x magnification. Orientation of the grids was always the same and the focusing was on t,he DNA or uranium oxide granules bnt never on tile latex spheres. (f) Quan,titative
analysis
of the micrographs
Electron micrographs were projected with 13 x enlargement and the contour lengths of tile DNA molecules, the RNA polymerase binding sites and the circumferences of the latex spheres were measured with a vertically mounted electronic graphic calculator with it Numonics Corp., Lansdale, Pa). The counter-balanced arm (Mark 3, 264-3605-136; measuring accuracy of this device was about t 0.5:,,, determined hp measuring the same object several times.
3. Results (a) I’isualization
of RNA
polymeruse
h&ding
E. coli RNA polymerase exhibits a general affinity for h DNA. but under the conditions employed (see Materials and Methods) only a limited number of polymerasr molecules is retained on A DNA (Fig. 1). The important features of our technique as applied to X DNA include: (1) use of glutaraldehyde at low concentrat)ion (O*lO,/,) to link preferentially to DNA the specifically bound RNA polymerase molecules, while inactivating those in solution ; (2) the dissociation of h DNA concatenates by formaldehyde treatment (at high concentration of 359;), in order to facilitate thtb gel filtration and to free the cohesive h termini, which served as primary reference points in the electron microscopic measurements; and (3) removal of the unbound RNA polymerase molecules by gel filtration on Sepharose 4B. It was necessary to apply the two-step aldehyde treatment (0.1% followed by 350/;,), as introduced by Vollenweider et al. (1976), since immediate exposure of the RNA polymerase-DNA complexes to the high concentration of formaldehyde results in the loss of most RNA polymerase bound, as also confirmed by Cherny et al. (1977). For DNAs which do not have cohesive ends, step (2) could be omitted (Koller et al., 1978). The globular molecules associated with A DNA in Figure 1 must be RNA polymerase, since such complexes were observed only when E. coli RNA polymerase was added. Also, the glutaraldehyde-pretreated E. coli RNA polymerase (0.104 glutaraldehyde, 10 min, 37°C) does not associate with DNA, indicating that only the polymerase already bound to DNA can be fixed by such treatment. (b) Mapping (i) Orientation
of RNA
polymerase
hindi,rg sites
and general mapping
Under the present conditions and with a polymerase-to-DNA molar ratio of about 30, up to 20 enzyme molecules are associated with each h DNA molecule, bound predominantly to one arm of the genome. Since most of the h promoters are located on the right arm, we assumed that, this is the arm which selectively binds RNA polymerase. and correspondingly we oriented the h molecules on the histogram shown in
488
H.
J.
VOLLENWEIDER
AND
W.
FIG. 1. Electron microgmphs of the E-. coZ% RNA polymerasc hyde/BAC technique, as described in Materials and Methods. preparations.
SZYBALSKI
bound to X 1)NA. The glutaraldr~. wax cmploy~tl in making thew
(a) 100 -
60-
60
70
80
90
IO
Per cent X length (“/.A mts) molrcule~ in relat,ion to the A genetic map. FIO. 2. Distribution of bound E. coli RNA polymerase Histograms (a) and (b) represent the left and right arms, respectively, of t,he entire h DNA molecule; (c) shows the refined mapping of the immunity and adjacent regions. The preparation of t,he RNA polymerass NDNA complexes is described in Materials and Methods. Employing the circummolecules were selected. The ference of latex spheres as a reference, only the full-lengt,h h DNA positions of the binding sites were mapped as the percentage of t,hr total contour length of each DNA molecule (in y0 h units). Polymerases bound to the DNA t,rrmini were not recorded. Each bar corresponds to 0*5% A (240 baw-pairs assuming the h penome is 48,000 base-pairs; Vollenweider et al., 1978), with the exception of the broken lines in (b) which correspond to 0.25% X. In histograms (a) and (b), 327 DNA molecules were analyzed and the ponit.ions of a total of 3448 bound RNA polymerases were measured in relation to the right A DNA terminus. In histogram (c), molecules in 83 DNA molecules were selected for analysis, each containing 2 or more polymerase t~he 77.5 t,o 81 o/o A region; t,he rightmost polymrrase in this region tiewed as the reference point (SW Results and Fig. 3). These 83 DNA molecules carried a total of 1075 polymerase molecules, with 366 of them in the mapped segment. The relative pwitions of histograms (b) and (c) reflect t,ho alignment of the statistical means of t,he 91.to-93:/; h ~wak in (h) and the corresponding peak in (c). On the genetic map, t,he approximate posit,ions and orirntations of the known promoters are respectively (see Hlattner et nl., 1974; Szybalski, 1976; indicated by the letter p and an arrow, Pilacinski et al., 1977). The symbols lit (si startpoint and t, terminator) and mis indicate short transcripts (Hayes & Szybalski, 1973; Carlson et CL, 1978; vitrd by Szybalski, 1976) and hip a site where new promoters can be created by mutations (Salstrom ut rtl., 1978). The leftward arrows in the 44.5 t,o 57.6O/, h region and the rlght,ward arrows in t,he 50 to 650/; h region correspond to the in vitro transcriptional starts init,iat,ed with pppA or pppG (Rosrnvold, Blattner & Hzybalski, unpublished data). The approximate positions of known terminat,ors are indicat.od by thr symbols t (see Szybalski, 1976; Pilacinski et rrl., 1977; Salstrom K- Sxybalski, 1978).
H.
490
J. VOLLENWEIDER
AND
W.
SZYBALSKI
Figure 2. In this histogram 327 intact X genomes associated with 3448 RNA poly merase molecules were analyzed. Figure 2(a) and (b) represents RNA polymerase binding to the left and right halves, respectively, of the entire X DNA molecules. The positions of the RNA polymerase binding sites were measured in respect to the right X terminus. The peaks correspond to the regions of most frequent polymerase binding, mainly in the center of the h genome and in several sites on the right arm. It is clear that under the present conditions RNA polymerase binds selectively to specific regions of the X genome. (ii) Background
of non-speci$c
binding
Very few polymerases bind to the left arm of X between 0 and 40% X, and since no known h promoters are located in this DNA region, we assume that these randomly bound polymerases represent the experimental background. If this background level is uniform throughout the entire h genome, we could calculate that under our conditions 16% of RNA polymerase binding is non-specific. (iii) Refined mapping
in the immunity
and adjacent regions
Since many important regulatory sites are located in the neighborhood of the h immunity region within the 70 to 90% X segment and several peaks of RNA polymerase binding are observed in this region (Fig. 2), a reference point located nearer to this region than the right X terminus was employed to shorten the measured distances and hence reduce the error. The position of the rightmost of two polymerase molecules (see Fig. 3) that bind to the segment between 775 and 81% X (Fig. 2(b)) was chosen as such a reference point, as indicated by the bar in the 79.5 to 80.0% interval (Fig. 2(c)). This procedure permits the resolution of several peaks in Figure 2(c), which appear to correspond to known X control elements in this region (see Discussion) and which were not discernible in Figure 2(b). (c) Analysis
of individual
RNA
polymerase binding regions
Examination of the histograms shown in Figure 2 reveals that RNA polymerase molecules bind to defined regions of the X genome represented both by the narrow and broad peaks of different heights. It is not clear, however, whether the broadness of a given peak indicates variability in the position of the binding site, inaccuracy of the measurements, or the presence of multiple and closely spaced binding sites. We therefore analyzed how many RNA polymerase molecules could simultaneously bind to each particular DNA molecule within the regions corresponding to the peaks. The results of this analysis, which comprised all the 327 DNA molecules represented in Figure 2 (with the 16% background of the non-specific RNA polymerase binding subtracted, as represented by the shaded areas), are shown in Figure 3. Some regions, as e.g., the 91 to 93% A segment, bind only one polymerase, whereas others, as e.g., the 44.5 to 58.5% X region, which comprises the A + T-rich b2 region, bind up to eight RNA polymerase molecules (Fig. 3, column 3). Column 6 represents the results of more refined mapping (Fig. Z(c)), which permitted resolution of the 77.5 to 8l*Oo/o X segment into two smaller regions, each binding only one polymerase. Similarly, the 69.5 to 77*Oo/o h segment was resolved into three separate regions. These results indicate that about half of the regions bind only one RNA polymerase for which a position can be statistically assigned (Table 1). The remaining regions
RN.4
POLYMERASE
BINDING
Number of RNA polymemses
TO
wlthin
Dato used in Fig. 2(a).(b)
COLIPHAGE
mdwduol
X DNA
491
bmdmg regkons
Doto used in Fig. 2(c)
I I
Uo. of polymeroses
No. of DNA molecules
region (%A) 1
60.0-69.5
1p
:
No. of polymeroses
No. of DNA molecules
I
I
P
5p
‘90
‘5,OI regbm (%A)
0
50
IOC
I
77*0-79,3 79.0
96.0-99.5
1;
1 I,
FIO. 3. Number of RNA polymerases within individual binding regions of h DNA. The binding regions (column 1) correspond to thr polymerase peaks on t,he X map in histograms (a) and (b) of Fig. 2. Each of the 327 A DNA molecules was analyzed individually, and the frequency of DNA molecules per region (column 2) is molecules simultaneously carrying 1, 2, 3 or more polymerase represent,ed in the form of separate histograms in column 3. The shaded portion of each histogram corresponds t>o thv experimental background of 16%, i.e. 5.5 polymerases per 1 o/0 A. The results in columns 4 to 6 arc presented in an analogous way using the data from Fig. 2(c), with the experimental background corresponding to 1.7 polymrrases per 19 h.
seem to correspond t’o clusters of two or more binding by th(x present, electron microscopic analysis. (d) Total number of RNA polyrnerase
sites that could not be resolved
binding
site.8 on h DNA
Twcsnty RNA polymerase molecules bind specifically to h DNA at a 30: 1 polymerase-to-h DNA molar ratio under the conditions used. This number is derived by adding the highest number of polymerase molecules bound in each region, after discounting the non-specifically bound polymerases indicated by shaded areas in column 3 of Figure 3. The number 20 remains unchanged when the data in column 6 are used.
492
H.
J.
VOLLENWEIDEH.
AND TABLE
W.
SZYBALSKl
1
RNA polymerase binding sites, promoters and some other regulatory &es on A DNA
Boundaries of the X DNA region ( y0 h)
No. of RN.4 polymcrase molecules bound wit,hin a region
44.5-58.5
x
58.5-62.0
2
68.0-69.5 70.3-71.8 72.8-73.8 73.8-75.8
1 1 2
77.8-79.3
1
794% 91.0-93.0 96.0-99.5
1 1 2
C’alculatrd position of the RNA pol,ymwasc? binding sitrt (O/” ,I)
Position and designation of t,hr h promoters, st,art,points and other transcriptioncontrolling elements (“/;, h)$ b2 rogion : two major 1 pppA startpoints two major I pppG startpoints one major T pppG startpoint 60.4; p, one T startpoint 68.6; hip, t,, il.1 ; f,, 73.5; ,‘L 75.7; lit, wis startpoint 74.3; ti 78.4; prm 78.5; *Ia 79.9; 1,” !12.3 ; p’*$ startpoirlt(s) OLL supcrhelical DN.4
h
The boundaries (column 1) were chosen to fit the valleys bet,wcrn the peaks. The number of RNA polymerase molecules (column 2) corresponds to tho highest nnmbcr in columns 2 and 5 of Fig. 3. t Statistical mean for all RNA polymerascs located in a given wgion. C’onfidenco interval calculated at 0.99 confidence level. i See legend to Fig. 2 and Discussion (section (c)). f Used as reference point in Fig. 2(c).
4. Discussion (a) XpeciJicity
of the glutaraldehyde/BAC
technique
When employing the glutaraldehyde/BAC technique, the specificity of RNA polymerase binding to X DNA was high, since only 160/, of non-specific binding was observed. In contrast, about 55O’,,0 of non-specific binding of RNA polymerase was seen in the absence of any fixatives or competing agents (Hirsh & Schleif, 1976). After our study was completed, a very similar result was also reported in the Appendix to the study of Williams & Chamberlin (1977). In their case use of 0.1 y0 of glutaraldehyde decreased the unspecific RNA polymerase binding by 35q& as compared with the glutaraldehyde-free controls. Our figure of 16% was derived by scoring the number of RNA polymerases on the left arm of X DNA, which does not contain any known transcriptional control elements. We recognize that the G + C content is higher in this arm than in other regions of X DNA ( see review of Davidson 8z Szybalski, 1971), but the base composition per se should not affect the RNA polymerase binding. For example, the residual and non-specific binding i?j, vitro of E. coli RlVA polymerase to the right arm (50-to-80%) region of the T7 genome is random (Port’mann et al., 1974; Koller et aE., 1978), although this DNA region contains distinct segments with high A + T content, as shown by partial denaturation (G6mez & Lang, 1972).
RN.4
POLYMEHARE
HIh’JlrNG
TO
COLJPHAGE
X IlRTA
4!K3
The reasons for the enhanced specificity may be manifold, but we shall consider two likely alternatives based on thth following schematic prewntations:
ONA t RP
I_ -2
ONA*RP
3 I +o
ONA
+ RP 4
4 1 +a
5
DNAmRIP P
-
6
“““‘97 P
If glutaraldehyde (g) reacts with free RNA polymerase (RP) reaction 3 results in inactivation of the enzyme (SW Results), which \\hen so modified cannot dissociate, into subunits (H. Bujard, personal communication). Reaction 4 is the first-step reaction between glutaraldehyde and RNA polymerase molecules bound to DNA. In case of non-specifically bound enzyme this fir&-step reaction may result in displacement of the polymeraac (reaction 5) since binding was weak (see Chamberlin. 1974). The second possibilit)y is that t’he enzyme would dissociate anyway (reaction 2 or 5) before t’he second-step fixation reaction 6 occurs, especially since the dwelling time of the non-specifically bound enzyme on DNA is short (see Chamberlin, 1974) as compared with the time required for react’ions 4 and 6 (Richards & Knowles, 1968 : Monsan et al., 1975). In the case of the specifically bound enzyme, the fixation (reaction 6) will br favored, sinw the dwelling time is much longer a,nd binding much stronger (see Chamberlin. 1974). To increase the specificity of RI\‘A polymerase binding for electron microscopic mapping, ot,her invest’igators used either poly( I) or heparin. Poly(1) appears less suitable since it permits about’ 25();, non-specific binding (data calculated from Fig. 5 of Hirsh & Schleif, 1976), whereas we observed only IS:;, for glutaraldehyde. It was not possible to estima,te the efft~ctiveness of hrparin from the data of Giacomoni ef nl. (1977). The frequency of RNA polymerase binding (nun] her of RNA polymerases visualized at each specific binding site, as shown in Figs 2 and 3) varies from one specific binding site to anot’her. Factors responsible for this variation (see scheme) may include (i) number of specifically bound RKA polymera,ses at the time of glutaraldehyde addition. (ii) the rates of dissociation (rewct)ion 2) and displacement’ 1)~ glutaraldehyde (r(‘action 5). and (iii) the rate of fixation (reactions 4 and 6). The rate of association (reaction 1) will become negligible soon after glutaraldehydc addit’ion since free RNA polymoraw will be inactivated b,v this fixative (reaction 3). Although we are not certain which of these factors an’ crucial in determining the frequency of RNA polymrrase binding to the h DNA, as visualized by the glutaraldehyde/BAC technique, thaw arc’ probably in common with t’hc fact#ors wsponsible for the retention efficiency of /\ DNA fragmcnt,s on nitrocellulose filtws in the presence of RNA polymerase and heparin (Jones ef ~2.. 19i7), becauw of the remarkable correlation between the rwults obtained by thew two methods (Fig. 4(b) and (c)). (I)) Comparison
with other methods of mapping
KflA
polymeruse
binding
sites
Early studies on RNA polymerase binding to h DNA led to the estimation of the total number of bound enzyme molecules, but not t’o their localization. For example.
H.
J. VOLLENWEIDER
AND
W.
SZYBALSKI
a)
b)
j-
:)
P
fmdll
I,
A&III
II
20
30
P
P
F
1
hdIIl IO
40
50
Per cent X length
60 (%A
70
00
90
I
mts)
FIG. 4. Comparison of the partial deneturation map of h UNA with the RNA polymerase binding map. (a) Partial alkali-denaturetion map kindly provided by Dr It. B. Inman and representing a refined version of the previously published map (Inman & Schniia, 1970) where t,he average deneturation per h molecule is 12.3%. (b) Map of E. co& RNA polymerese binding &es derived from Fig. 2 (a) and (b) with the experimental background subtracted. (c) Map of the (i) Hind11 and Hind111 cleavage sites represented by the downward or upward pointing arrow, respectively (Robinson & Lendy, 1977a,b); (ii) Hind11 and III fragments that bind E. coli RNA polymerese and are efficiently retained on filters at an 11: 1 polymerase-t,o-h molar ratio (black rectangles), inefficiently retained (shaded rectangles), or retained only at a 47: 1 molar ratio (open rectangles), when X DNA and RNA polymerese were incubated for 30 min at 37°C and then treated with an excess of heparin for 10 min at 37°C (Jones et nl., 1977); and (iii) the cleavage sites protect)ed by the bound RNA polymerase as indicated by symbol P (Jones et crl., 1977). Since XpZnc5 was used in these experiments, the region within brackets does not correspond to maps (a) and (b).
Nakano $ Sakaguchi (1969) reported that up to 18 polymerase molecules could bind to h DNA, in good agreement with the present results. Fragmentation of DNA by the restriction nucleases permitted the assignment, of RNA polymerase binding sites to those X fragments retained on nitrocellulose filters. The results of such a study by Jones et al. (1977) are shown in Figure 4(c). It can be seen that there are at least nine fragments that bind polymerase, four of them with the highest efficiency and two with the intermediate one. The location of those fragments on the h map and the efficiency of their retention (measured as the required polymerase-to-DNA ratio; Fig. 4(c)) a g ree well with our data as represented by the positions and heights of the peaks (Fig. 4(b)). It might be interesting to note that some fragments were retained on the nitrocellulose filters only at RNA polymerase/h DNA ratios as high as 47 : 1 (open rectangles in Fig. 4(c)), whereas we could visualize same RNA polymerase binding at probably the same sites already at a ratio of 30 : 1.
KS.1 POLYMEHAHE
BINT)ING
TO C'OLlf'H;\GE
A DSA
Other data, based on the protection of restriction sites and initiation synthesis, will be considered in the following sections. (c) Comparison, of the positions of the RiVA polymerasp with those of the known promofers
binding
495 of RNA
sites
The binding of RNA polymerase to X DNA. as discussed up to this point, did not imply any biological funct~ion for this interaction. However, it is known that RNA polymrrase binding is one of the steps in the initiation of t’ranscription, and t,he availability of detailed maps of t,he in viva and i,r vitro initiation sites in h permits the determinat,ion of which of the binding sites represent t,he active promoters. The positions and orientation of the known promoters are indicated by the horizontal arrows for the left (Fig. 2(a)) and right arms (Fig. 2(b) and (c)) of h DNA. Witlr few exceptions the correlation between the peaks indicating the binding regions and the positions of the promoters is very good. On the left arm there are no peaks of specific ROTA polymerase binding and there are no know,n promoters (Fig. 2(a,)). The first two peaks between 44.5 and 585°{I /\ correspond to several leftward and right ward startpoints observed during 20 seconds of ire vitro transcription in t,he A + Triclr hd region of h, as determined by Rossnvold: Hlattner & Szybalski (unpublished data). Their lower estimat’e for t,he number of strong startpoints is five. whereas wc observe eight binding sites in this region (Fig. 3). Proceeding rightward. a small peak of RN,4 polymerase binding could be observed to two binding sites (Fig. 3), in the 58.5 to 62.0% A region (Fig. 2(b)). corresponding which could represent the ~pr promoter located at 60.4’$; /\ (Honigman & Hu? 1978: Pilacinski et aZ., 1977) and a righhward start observed in rrifro by Rosenvold. Blat’tner & Sz,vl)alski (unpublished data). The major pL and pR promoters correspond to the single binding sites at 73.3 and (Fig. 2(b) and (c), and Table 1). The p0 promoter for 0023RNA 78.4”,‘, /1, rrspect,ively corresponds to the single binding sit’e at 79.8 9,;)h. The agreement between our measurements and the sequencing data is excellent since we measured the distance between are 653 base~1~and p,, as 650&35 base-pairs. whereas t’he corresponding startpoints pairs apart according to sequencing data of Schwarz et al. (1978). We saw only one polymerase binding site in the ~I,.~-P~ region and the simplest’ interpretabion is that under our conditions only very few or no RNA polymerases bind to the p,., promoter. When st,udying the protection of the prm and pR sites from nucleolytic digestion under comparable conditions (Meyer et al.. 1975), B. Meyer (personal communication) observed RNA polymerasc binding to the p,, promoter only when t,he ratio of polgmerase to the prrn promoter was ovtr 20. whereas in om case it \vas less than 2 (30 polymerases per 20 binding sites). We observed two polymerasc binding sit)es located to t’hr left of yL, one at 68,6 and another at 71.00,:, h. The tirst one corresponds either t’o t,he position of the t,, leftward terminator or to the site where point’ mutations could easily generate a new left(ward promoter, designated hip (Salstrom & Szybalski, 1978: Salstrom et al., 1978). The second. at 71.0% X corresponds to the position of the t,, leftward terminator site (Salstrom & Szybalski, 1978). We do not know ðer t’he present’ly observed RKA polymerase binding near or at the terminators could have some role in the termination function. It is unlikely that strong polymerase binding should be a general feature of the terminat,ors. since we did not observe any strong binding at the site corresponding
H.
496
J.
VOLLENWEIDER
AND
W.
SZYBALSKI
to the third strong leftward terminator, t,,, located at 64.4% h (Salstrom t Szybalski, 1978). We observed two binding sites within the middle of the h immunity region at 73% to 75.8% h, but their relationship to the startpoint (sr) or terminator (ti) of the in viwo produced lit transcript located between 74.3 and 75.6%) \ (Hayes & Szybalski, 1973) or to the in vitro-synthesized m,is transcript (Carlson et al., 1978 and cited by Szybalski, 1976) is unknown. The single binding site at 92.0&O-l “/o h (Table 1) shows excellent correspondence to the position of the startpoint controlled by the pk late promoter calculated to be at 92.3% X as based on the data of Sklar et al. (1975) and Robinson $ Landy (1977a,b). The remaining two binding sites in the 96.0 to 99*50/b h region do not correspond to any known X control sites that operate in vivo. However, $llet & Solem (1974) detected RNA polymerase binding in this region which protected one site from Hind11 endonucleolytic cleavage (see also Jones et al., 1977), and Botchan (1976) has been able to force initiation of in vitro transcription in this region by using supercoiled circular h DNA templates. The latter data do not permit one to distinguish between one or more transcriptional startpoints in this region. The data discussed in this section are summarized in Table 1. (d) The number of sites, the frequency of RNA polymerase bind&g the eficiency of in vitro transcriptional starts
sites and
In several studies the initiation of transcription was used as a method for scoring the polymerase binding sites on h DNA. For example, Nakano & Sakaguchi (1969) observed 17 sites on which RNA polymerase initiates transcription and Willmund & Kneser (1973) estimated that there are six to 11 or more such sites. Furthermore. data listed in Table 1, which indicate eight known transcriptional starts on t,he Xl12 template (i.e. outside the 62 region). together with the results of Rosenvold, Blattner & Szybalski (unpublished data) showing live startpoint’s in the b2 region, add up to 13 polymerase initiation sites. Thus, the number of active initiation sites seems to be somewhat lower than the total number of binding sites observed in our study (Table 1). In Table 2 we compared the frequency of polymerase binding at the sites corresponding to the four known promoters, (I)~, pR, y0 and pa, with the efficiency of initiation of in vitro transcription at these promoters, as calculated by Dahlberg & Blattner TABLE 2 Frequency
of RNA polymerase bindkg and eficienc?/ of in vitro trarwription at Jive binding sites on X I)NA RNA
0/0 binding of RNA polymeranet T/, in vitro initiation of RNA synthesis by RNA polymerase$
polymcrase
binding
PI.
1’11
PO
21 22
2I 24
16 (i
Numbers in each line add to 1000/b. t Derived from data in Fig. 2. $ Based on data of Dahlberg & Blattnw
(1973)
for linear
sites and promoters 9G-99.5% PK 20 48
h DNA.
initiation
oj
X region ‘3
RNA
POLYMERASE
BINDlNG
TO
COLIPHAGE
h DNA
497
(1973). The correlation is very good for the p, and pR promoters, but there is a morel than twofold difference at the p, and & promoters. No initiation of transcription in the 96 to 99.50,/, h region was reported by Dahlberg & Blattner (1973), huh as already mentioned, Botchan (1976) was able to force in vityo transcription in this region on a supercoiled DNA template. The present results show that) supercoiling is not required for an ef?icient binding of RNS polymerase, but it is not clear whether it aids in the initiation of transcription or abolishes some closc~ly associated termination event. (e) Relationship
betwee?%the pol?ymerase hindirly
sites ad
A + T-rich
regions
There appears to be a remarkable correlation between the position of the RR’A polymerase binding sites and the A + T-rich regions, the latter determined by partial denaturation mapping (Fig. 4). Similar conclusions were recently reached both for h DNB (Jones et al., 1977; Botchan, 1976) and for coliphage Ml3 DNA (Dasgupta et ad.. 1977). Close inspection, however, reveals an imperfect quantitative correspondence for a few regions. For example, the ratio of peak heights in regions 91 to 93 and 96 to 99.5s/, h in Figure 4(a) is inverse to that in Figure 4(b). Some of the most prominent peaks of polymerasc binding and denaturation bubbles are not associated with any known in vivo acting promoters, alt’hough in vitro initiat,ion of transcription was observed in these regions (see regions 44.5 to 58.57; h and 96 t,o 99*5’:/, ,I). Furthermore, F. coli RNA polymerasc? does not bind to d + T-rich regions per se. as already discussed for the T7 phage genornc (Keller et al., 1978: G6mez & Lang, 1972). These data strongly suggest that high A--T contcant is an important but not sufficient criterion for specific and &Gent initiation of transcription.
We t,hank Mrs A. James for excellent technical assistancc~. 111’ R. B. Inman generol~sl> supplied us with the most current denaturation map of A DN,l (Fig. 4(a)) and Dr R. 1~. Burgess with the E. coli RNA polymerase. lsTe thank Drs R. R. Burgess and T. Koller for critically reading the manuscript and Dr E. Szybalski for editorial help. We also express our appreciation to Lois Wilson and
REFERENCES Allet, 13. 85 Solem, R. (1974). J. ~21ol. Biol. 85, 475-484. Blatt,rler, F. R. & Dahlberg, J. E. (1972). lVa,ture Nev* .Gol. 237, 227-232. Blattner, F. R., Dahlberg, J. E., Boettiger. J. K., Nantit, M. & Szpbalski. W. (1!)72). Nature New Biol. 237, 232.-236. Blattner, F. R., Fiandt, M., HRSS, K. R., Tn-osr. 1’. A. S- Szyba,lski, W. (1974). l’irolo~gy, 62, 458-471. Botchan, I’. (1976). J. &foZ. Bid. 105, 161-176. Bsvre, K., Lozeron, H. A. & Szybalski, W. (I 971). In &‘ethods in I~irology (Maramorosch, K. & Koprowski, H., eds), vol. 5, pp. 271-292, Academic Press, New York. Carlson. K., Krill, J. & Szybalski, W. (3978). illul. Gen. Gevet. in tlw press. Chambcrlin, M. J. (1974). Ann~u. Rev. Biochem. 43, 721-775. Cherny, D. I., Alrksandrov, A. A., Zarudnaya, M. I., Kosa~anov. ‘I’. N., Lazurkin, Y. S., Bmbealashvilli, R. S. & Savochkinn, L. I’. (1977). Eur. .I. Biochem. 79, 309-317. Dahlbera, .J. E. & Blattner, F. K. (1973). In I’imrs Resenrch, 2nd 1. c:. N.-U.C.L.A. Syrrrp. on Mol. Biol. (Fox, (:. F. & Robinson, TV. S., vtln). pp. 5X&543, Academic Press. New York.
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VOLLENWEIDER
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W.
SZYBALSKI
C. E. Kt Mitra, S. (1977). .J. BioZ. Chem. 252, Dasgupta, S., Allison, D. P., Snyder, 5916-5923. Davidson, N. & Szybalski, W. (1971). In The Bacteriophage h (Hershey, A. D., ed), pp. 45-82, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Giacomoni, P. U., Delain, E. & LePecq, J. B. (1977). Eur. J. Biochem. 78, 205-213. G6mez, B. & Lang, D. (1972). J. Mol. Biol. 70, 239-251. Hayes, S. & Szybalski, W. (1973). Mol. Gen. Genet. 126, 275-290. Hirsh, J. & Schleif, R. (1976). J. &foZ. Biol. 108, 471-490. Honigman, A. & Hu, S.-L. (1978). IT’iroZogy, in the press. Inman, R. B. & SchnGs, M. (1970). .J. Mol. BioZ. 49, 93~.98. Jones, B. B., Chan, H., Rothstein, S., Wells, R. D. & Reznikoff, W. (1977). Proc. Nat. Acad. is., U.S.A. 74, 4914-4918. Koller, T., Sogo, J. M. & Bujard, H. (1974). Biopolymers, 13, 995-1009. Koller, T., Kiibler, O., Portmann, R. & Sogo, J. M. (1978). J. f!oZ. Biol. 120, 121-132. Maniatis, T., Ptashne, M., Backman, K., Kleid. D., Flashman, S., Jeffrey, A. & Maurer, R. (1975). Cell. 5, 109-113. Meyer, B. J., Kleid, D. G. & Ptashne, M. (1975).Proc. h’at. Acad.Xci., U.S.A. 72,4785- 4789. Monsan, P., Puzo, G. & Mazarguil, H. (1975). Biochimie, 57, 1281-1292. Nakano, E. & Sakaguchi, K. (1969). J. Biochem. 65, 147-150. Pilacinski, W., Mosharrafa, E., Edmundson, R., Zissler, .J., Fiandt, M. & Szybalski, W. (1977). Gene, 2, 61-74. Pirrotta, V. (1976). In Current Topics in Microbiology ad Immunology, vol. 74, pp. 21 -54, Springer Verlag, Berlin. Portmann, R., Sogo, J. M., Koller, T. & Zillig, W. (1974). FEBS Letters, 45, 64.-67. Ptashne, M., Backman, K., Humayun, M. Z., Jeffrey, A., Maurer, R., Meyer, B. & SRIIPI., R. T. (1976). Science, 194, 156-161. Richards, F. M. & Knowles, J. R. (1968). J. Mol. BioZ. 37, 231-233. Robinson, L. H. & Landy, A. (1977a). Gene, 2, l-31. Robinson, L. H. & Landy, A. (1977b). Gene, 2, 33-54. Salstrom, J. S. & Szybalski, W. (1978). T’irology, in the press. Salstrom, J. S., Fiandt, M. bi Szybalski, W. (1978). Mol. *en. Genet. in the press. Schwarz, E., Scherrr, G., Hohom, G. & Kiisscl, H. (1978). ilrature (London), 272, 410-414. Sklar, J., Yot, P. & Weisman, S. M. (1975). I’roc. Nat. Acad. Sci., U.S.A. 72, 1817-1821. Szybalski, W. (1976). In Handbook of Riochemhstry and Molecular Biology (Fasman, G. C., ed.), 3rd ed., N,zlcZeic Acids, vol. 2, pp. 677-685, CRC Press, Cleveland, Ohio. Szybalski, IV. (1977). In Regulatory Biology (Copeland, .J. C. & Marzluff, G. A., ods), pp. 3-45, Ohio Sta,te University Press, Columbus, Ohio. Vollenweider, H. J., Sogo, .J. M. & Keller. T. (1975). I’roc. Nat. Acarl. Sci.. T7.S.B. 72, 83-87. Vollenweider, H. J ., Koller, T., W eb er, H. 85 Weissmann, c’. (1976). ,J. ~%fol. Biol. 101, 367-377. Vollenweider, H. J., James, A. R: Szybalski, W. (1978). I’roc. Nat. AC&. &i., I.S.A. 75, 710-714. Westmoreland, B. C., Szybalski, W. & Ris, H. (1969). Science, 163, 1343-1348. Williams, R. C. $ Chamberlin, M. J. (1977). Proc. Nat. Acad. Sci., D.S..4. 74, 3740-3744. Willmund, R. & Knoser, H. (1973). ,?foZ. Gen. Genet. 126, 165-175. Note added in proof: Recently (W. Taylor and N. Burgess, unpublished 6.916 fragment (93.19 to loo?/,) situated to the right. of pa’ (Sklar shown to behave like other h promoter-containing fragments. It polymerase and is retained on a nitrocellulose filter along with other fragments. In addition, when ribonucleoside 5’-triphosphates are reaction, initiation can occur so that this fragment (like other fragments) is retained on the filter even after a 0.6 &r-KC1 wash.
results) the /\.%oRl et al., 1975) has been binds h’. coli RNA promoter-containing added to the binding promoter-containing