J. Mol. Biol. (1976)
101, 367-377
Physical Mapping of Q/? Replicase Binding Sites on Q/? RNA H. ,T. VOLLENWEIDER, TTI. KOLLER Institut fiir Zellbiologie ETH- Z, Hiinggerberg 8039 Ziirich, Su&erland
H. WEBER AND CH. WEISSMANN Imtitut
fiir Molekdarbiologie 1 Universitdt Ziirich NAdt~pentlrnt, RNA polymerase of E. coli per ml. (2) Tlrca saln(~ RN.4 ant1 RN.4 polynlc~lW concentrations as in (1) but otherwise under the conditions used for the binding of E. co/i 8 rnx-magnesium RNA polymerase to T’7 DNA (Portmann et al.. 1974). i.e. 50 rnM-KU, nc*ctatc and 30 mM-triethanolamine-HCl at pH 7.9. \vith incubatiorl for 10 min at 37-(‘.
(e) Stabilization
of th.e complexes
and
tle~twtrwation
qf the
HSJ
iI1 80 m>,To 100 ~1 of incubation mixture were added 20 ~1 of 1 Ijo forlnaldehydo trirthanolamine-HCl at pH 7.5. After heating to 37°C’ for 10 mirl, 880 ~1 of 3.5O, formaltl~hydt: in 80 mM-triethanolaniine-HCl (pH 7.5) were adtic~l. Tllc tc~lnpt~raturt~ uxs t Il(srl raised to 63°C for 20 min (Boedtker, 1971).
(f) Spreading
of the comp1e.re.s
The procedure followed was exactly as described by Vollerl\l,cidet, et al. (1975), rsctspt tllat ttle stock solution of BAC was diluted in a buff~~r containing 3.54; formaldcllydo and 80 m&I-triethanolamine-HC1 at pH 7.5. Spreadin,c was pcrforrncd ont,o a Itypopllasrb of frctslil>- redi&illod water.
(g) Preparation.
of specimens
.for th,e electron,
m,icroscol)e
The RNA was picked up onto 400-mesh grids co\-errd wit,h standard carbon films (film t,hickness about 90 d as est,imated by a Balzers quartz crystal tllin film monitor). The specimens were washed by floating on redistilled wat)er for IO min. They were thctl dehydrated in 90% ethanol and stained by dipping int,o a frctshl\- prepared solution of 900,& &hanol, 1 In&r-uranyl acetate, 1 mM-HCl for 10 s. The excess staining solution \VRS removed by briefly washing the specimen in 9O”/b rthanol and drying it on filter Papa. Finally the grids were rotary shadowed at an angle of 7’ from an electron gun (Moor. 1970) with 1000 Hz carbon-platinum, as measured on the thin film monitor.
(h) Electron
microscopy
A Siemens Elmiskop 101 electron microscope operating at 100 kV accelerating volt,apcL was used with a 400 pm aperture in the second condenser lrns and a 50 pm aperture in the objective lens. Micrographs were taken at 27,000 x electron optical magnification as determined on a carbon grating replica from Balzers Union, Liechtenstein. The variation in rnagtlification for different specimens was 30/:, or lcsa (li. Portmann & .J. Hogo, rlnpul~lishctl rcsult,s). Micrographs wore recorded on Kodak c+lnrtror~ image plates tl~~~~clopt~~t :I( 20’ C’ f’or 4 min ill Kodak HRP dc:\-eloper (dillltiorr I : 4).
FIG. 2. Electron micrographs of Qb RliA-mplicase comploxaa. The preparations are described in Materials and Methods. Magnification: 110,000 x . A, 811 extended complex of Q/3 replicase and Q/J RNA. B and C, looped complexes of Qfi replicase and Qj3 RNA. Note the replicaso molecule at the base of the RNA loop.
Qb REPLICASE
BINDING
(i) Quantitative
SITES
ON
Q/3 RNA
371
analysis of the micrographCs
Selected molecules were printed at 10 x enlargement. Length measurements were et aE., 1974). The made with a Hewlett-Packard digitizer as described earlier (Portmann measuring accuracy was 0.75 mm, corresponding to O-003 Frn at the final magnification. The class sizes of the histograms wore 0.04 pm (Fig. 3) or around 0.016 pm (l%, Figs 4 and 6).
3. Results It has been shown earlier that incubation of Qp RNA with Qp replicase under conditions of RNA synthesis but’ in the absence of GTP and host factor I to preclude initiation, leads to formation of a stable complex which initiates RNA synthesis upon addition of the missing components (H. Weber, unpublished results). Such complexes first’ at low formaldehyde concentration at were fixed in a two-step procedure: 37”C, to stabilize the protein-RNA complex, then at higher formaldehyde concentration, at 63”C, to denature the RNA. Under the electron microscope the stained and shadowed preparations showed extended filaments. 60% of which were associated with a single blob. Among bhe strands with blobs two characteristic forms were
64
0 25
05
0.75
IO
Distance (pn) FIG. 3. Histograms of the location of Qp replicate binding sites on Q/3 RNA. The preparation of the complexes is described in Materials and Methods. Abscissa: distance of enzyme molecules from the RNA end. (a) 44 extended complexes of Qfl replicase and Q/3 RN.4. The end of the shorter RNA segment was positioned at the origin of the histogram. (b) 62 looped complexes of Qfi replicase and Q/3 RNA. For each complex, the lengths of both the longer and the shorter branches were plotted separately (differentially hatched areas). The black outline represents tho summation curve of the 2 individual distributions.
1172
Jf.
.I.
\'01,1,I~:Z\2~EII~E:li
KY'
.-I I,.
ol~sr~rved : ( I ) OIW third comprised fully extended filaments u:ith >\ single I)lol~ loc:atwl at around 300,, from one end of the molecule (Figs 2A and 5) two thirds \IW(’ strands with a loop and a blob located ;I,t the ba,se of t’hr> loop (Fig. %H and a(‘). Since in a11 wntrol exprrimrnt~s in \j.hich replicase was omitt,cd such I)lohs \\ VU~ tlxtremely rare and were not, observed in conjunction with a ~OJ) (in no castL among 350 filaments inspected). MY considered them to represent Q/3 wplicaw moleoult~s l)!)und to Qp R’NB. Accordingly. \ve will henceforth call thrw blobs wplicasc* tnolmllw.
The proportion of broken strands was variable in different experimctnts. possii)lJ due t*o differences between individual RNA batches as well as to variations in spreading. We t’hereforc selected the longest strands for the quantitative analysis (abow I + pm) a,nd considered these to be full length Q/3 RNA molecules. The longest strands with a bound replicase molecule \I.ere 1.7 pm. Qfl REA wit,hout replicaw. measured on (~xprriment,al or on control grids. showed strands with a length of up to 1.9 pm. Previous cbxperimcnts hare shown that after treatment of Q/3 RNA-Q/3 replicase complexes with T, RKase RNA fragments corresponding to about 350 nucleotides are retainc~tl by. C&3 replicase (Meyer. Weber & WFeissmann, unpublished results). It is possible. but difficult to ascertain, t)hat the apparent short,ening of t#he RNA by 0.2 pm (ahout 470 nucleotid&) is due to the coiling of RR’A ont’o thc& enzyme’. WV first analysed the complexes wit,11 110 RNA loop and mapped the positions of the rcplicase molrcules. In no case did we find a complex which had more t’han one wplicase nloleculc associated with the RPL’A. This \\.a~ also bhe (;ase \vhen \vt: increased the enzyme-t)o-RNA ratio I)y a factor of ten. compared to the standard procedure described in Materials and Methods, to give a molar ratio of about, 30 : 1. At a moln,r enzyme-t,o-RNA ratio of about 3:l some blobs not associat)ed with RSA were observed: the amount, of unbound blobs \vas substantially greater at a molar rat,io of 30 : 1, Figure 3(a) gives the histogram of the location of 44 replicaw molecules on t,hc R&A. The strands ww oriented such that the end of the shorter A rela,tivel>, hoad region wgment, was positioned at the origin of the histogram. of replicaw binding sites around 0.5 pm was obtained.
10 Qp RNA mop units p/o)
Fro. 4. Histogram of Q,9 replicesc binding sites on Q/3 RNA in looped complexrs. The preparat~ion of the complexes ia described in Mat,erials and Methock. Abscissa: position of binding sita in map units (the length of each RNA molecule was set to 100%). 62 complexes wvw analysrtl. The end of t,he shorter RNA branch was placed at the origin of t)he histogram.
374
H.
J.
VOLLENWEIDER
E!Z’
:iL.
The location of the replicase binding sites on the RNA of the looped complexes is shown in Figure 4. Again we positioned the end of the shorter RN,4 branch at the origin of the histogram. Since only 6% of the RNA strands without a bound replicast: molecule showed any kind of a loop (among 350 strands inspected) and since in Hit! looped complexes the replicase molecules were located always at the base of the loop, we conclude that the appearance of the loop was due to interaction of Q/3 replicaso with two sites on the RNA. The mean positions of these sites were at 0.46&OGl pm and at 1.03+0.07 pm from the end of the shorter RNA branch. In order t’o eliminate effects like variations in electron optical magnification or different extent of stretching of the RNA during spreading, we normalized the data, taking the length of each individual RNA molecule as 100%. The binding sites thus obtained \\ere located at. 29-5&2*5o/o (map units) and at 65f3.5% from one end. In Figure 3(b) we plotted the lengths of both branches of the looped molecules. The mean values for the two peaks in the histogram thus obtained were 0.46+0.04 pm (the same as described above) and 0.55kO.06 pm. No replicase associated with RNA could be observed under conditions which are specific for binding to only one of the two sites (Weber et al., 1974). As a control to these experiments \ve incubated E. coli RNB polymerase with Q/I RNA under our standard replicase binding conditions (Fig. 5A) as well as under conditions optimal for the binding of this enzyme to T7 DNS (Portmann et al., 1974) (Fig. 5B). Several (up to 5) enzyme molecules were bound to each RNA strand and their distribution along the Q/3 RNA strand was clearly random, except for some selective binding of enzyme molecules to termini (Fig. 6; orientation of the strands was again such that t#heend closest to a polymerase molecule was at the origin of the histogram). Only in 7% of the complexes was there a loop, of variable size, associated
Qp RNA map units P/d
FIG. 6. Distribution of DNA-dependent RNA polymer5se molecules bound to Q/l RNrl. The complexes were prepared as described in Materials end Methods. Abscissa: position of binding sites in map units (the length of each RNA molecule was set to 100%). The end of the RNA closest to a polymerase molecule was placed at the origin of the histogram. Blank colwnns: Black columns : binding coru s&me incubation conditions as for RNA-&B replicase complexes. ditions used by Portmann et al. (1974) for DNA-dependent RNA polymerese of h’. COG.
Q/3 REPLICASE
BINDING
SITES
ON
Q/3 RNA
375
with the bound enzyme. These findings are in agreement with the observation that when complexes of RNA polymerase and Q/3 [32P]RNA were treated with RNase T, the RRA fragments retained by the enzyme could not be resolved into discrete bands by polyacrylamide gel electrophoresis, which also suggested that binding did not, tske place at specific sites of the Q,4 RNA (data not shown).
4. Discussion We should emphasize that for technical reasons the data used for obtaining the hist,ograms could not be collected in a random fashion. Only those regions of the preparation could be analysed where the uranyl acetate st’aining was smooth and clean. and where the carbon-platinum shadowing gave a good signal-to-noise ratio. Furthermore, a variable number of RNil molecules was broken, making a rigorous selection of the longest strands necessary. Since the selection had to be done on several levels (staining, shadowing, association with enzyme, RNA length), it was impossible to estimate the yield of complexes which could be used for the mapping of replicase binding sites. The maximal length of uncomplexed Qp RNA reported in this work (1.9 pm) is clearly greater than has been described earlier (Koller rt aZ., 1971, 1.14kO.12 pm; Yuh Chi & Bassel, 1974, l*ll&O.ll pm; Sogo et al., 1974, 0.83kO.14 pm). This increased length of single-stranded nucleic acid molecules spread with the BAC method has already been observed with denatured T7 DNA (Vollenweider et al.. 1975). Under the assumption that unbroken Qfi RNA contains about 4500 nucleotides (Boedtker, 1971; Reijnder et al.? 1973; Yuh Chi & Bassel, 1974), we calculate for the spreading conditions of the present work an internucleotide distance of the order of 1 ,r(. It is not clear why Q/3 R1L’A complexed with Qp rnplicase appears to be shortened 1)~ about 0.2 pm. It is not possible to correlate the molecular weight of the replicase molecules with their apparent diameter since the latter depends on the amount of stain bound, which is undetermined and variable under the conditions used (Zobel $ Beer, 1961). The present work has shown that two thirds of the Q/l RNA-Q/I replicase complexes observed under the electron microscope contain an RNA loop with an enzyme molecule attached to its base. The loop constitutes about 3596 of the genome length, while the two free branches comprise about 30% and 350,x of the length, respectively. showed loops, and these Only 6$‘{, of the free RNA strands in the same preparations were of varying size and at diverse locations. Studies designed to reveal the secondary structure of Q/3 RNA (A. Jacobson & H. J. Vollenweider, unpublished data) shorn tha,t under less rigorous denaturing conditions a, small loop (about 0.1 pm in size) regularly occurs very close t’o one end, while loops as seen with the replicase-RNA complexes could not be found. We therefore believe that the loop in the complex comes about by the crosslinking of one Qp replicase molecule to two distinct sites of the RKA which are linearly far removed from each other, but, may of course be spatially adjacent in the native molecule. We have never observed RNA filaments to which two replicase molecules were bound, even when the molar ratio of enzyme-t,oRNX was increased tenfold to 30. This is in contrast to the control experiments with DNA-dependent RNA polymera,se, where several. np t)o five, molecules were bound t,o one Q/3 RNA. This finding is compatible with t,he assumption that Q/? replicase binds simultaneously or in rapid succession to both sites to form a stable looped complex, and that t,he extended complexes with one bound enzyme come about by
37Ci
r-I
.I
1.0 1, I, b: N \\’ E I I ) E I< E 7’
I 1.
parCal disruption of bhe looped complex during tixatiott or spreading. ‘1’11~ I1istjogratil of Figure 3(b) shows the length distribution of \wt,h 1)ranches of eaclt of the I00ped trtolccules. The tn.0 result,inp peaks overlap partially and if t,he suttttttation wrv~ is drawn (see Fig. 3(b)), one obt,ains a distrihubioti which resemhlrs the histogram fot the cxtendcd complexes (Fig. 3(a)). l’hcrefow. in t~lte extended c~m~plexes. tltc: r~plicase could 1)~ atItached to one or the, ot,hw site with al)out ~~(luitl frqu~~nv~~. Tlte observation. that under conditions where biochemical experitttettts Itav(B demonstrated stable binding to only one site no complexes are found. tnight also he explained by disruption of the complexes during the preparation. ‘I’he electron microscopic ohservat,ions are compatible witlt our previous I)iochemical findings. It \\‘as shown earlier t)hat if [“‘TJQ@ RNA-Q/3 replicase complrxcs, formed under the same conditions employed in the present work. wew treated with RNase T,. several RNA segments remained hound t,o t’hr enzyme. One 1at.g~ RNA piece (fragment S-3. 100 nucleot~ides) was sho~vtt to be derived from the region preceding the coat, cistron. i.e. ahout 31”,, of the genotne length from the 5’ end (Weher of al., 1971). while three othtbr fragments (M-2. M-5. and M-I 1. \rith 164. 60. and 21 nucleotides. respectively) probiibly originat,ed from a region Lvit(hin the twginning of t)he replicase cistron. about 47 t,o A()“,, of t#he gcnomc tcnpth from the 5’ end (Weber rt rrl.. 1974; Meyer rf 111.. 1975). The position of t)he S site. which could he located fairly accurately on the genomc (cf. Fig. 1). is pract,ic:ally identical wit,h that of one of the binding sites identified by elect,ron microscopy. The position of t,hr other site, as det’ermined I)y electron tnicroscopy, falls just outside the region from which the M fragments are believed to originate. Since the biochr:mical mapping of the M site was not ver) ibccuratt~ we believe t’hat our data are contpat,iItle \qith the t\vo positions being identical. Alt~crnat.ivcly, replicasisslnann. C.. Billeter, M. A., (inodman. H. M., Hindley. ,J, & Weber, H. (1973). *-Jr~lol. ICer.
Hiochem.
42,
303-328.
Yuh (‘lli. Y. & Bassel, A. R. (19i4). J. Viral. 13, 1194 1199. %oi)cal. (‘. Ii. h Hfbrr, M. (1961). J. Riophys. Hiochem. Q/to/. 10. 333
346.
101, 367-377
Physical Mapping of Q/? Replicase Binding Sites on Q/? RNA H. ,T. VOLLENWEIDER, TTI. KOLLER Institut fiir Zellbiologie ETH- Z, Hiinggerberg 8039 Ziirich, Su&erland
H. WEBER AND CH. WEISSMANN Imtitut
fiir Molekdarbiologie 1 Universitdt Ziirich NAdt~pentlrnt, RNA polymerase of E. coli per ml. (2) Tlrca saln(~ RN.4 ant1 RN.4 polynlc~lW concentrations as in (1) but otherwise under the conditions used for the binding of E. co/i 8 rnx-magnesium RNA polymerase to T’7 DNA (Portmann et al.. 1974). i.e. 50 rnM-KU, nc*ctatc and 30 mM-triethanolamine-HCl at pH 7.9. \vith incubatiorl for 10 min at 37-(‘.
(e) Stabilization
of th.e complexes
and
tle~twtrwation
qf the
HSJ
iI1 80 m>,To 100 ~1 of incubation mixture were added 20 ~1 of 1 Ijo forlnaldehydo trirthanolamine-HCl at pH 7.5. After heating to 37°C’ for 10 mirl, 880 ~1 of 3.5O, formaltl~hydt: in 80 mM-triethanolaniine-HCl (pH 7.5) were adtic~l. Tllc tc~lnpt~raturt~ uxs t Il(srl raised to 63°C for 20 min (Boedtker, 1971).
(f) Spreading
of the comp1e.re.s
The procedure followed was exactly as described by Vollerl\l,cidet, et al. (1975), rsctspt tllat ttle stock solution of BAC was diluted in a buff~~r containing 3.54; formaldcllydo and 80 m&I-triethanolamine-HC1 at pH 7.5. Spreadin,c was pcrforrncd ont,o a Itypopllasrb of frctslil>- redi&illod water.
(g) Preparation.
of specimens
.for th,e electron,
m,icroscol)e
The RNA was picked up onto 400-mesh grids co\-errd wit,h standard carbon films (film t,hickness about 90 d as est,imated by a Balzers quartz crystal tllin film monitor). The specimens were washed by floating on redistilled wat)er for IO min. They were thctl dehydrated in 90% ethanol and stained by dipping int,o a frctshl\- prepared solution of 900,& &hanol, 1 In&r-uranyl acetate, 1 mM-HCl for 10 s. The excess staining solution \VRS removed by briefly washing the specimen in 9O”/b rthanol and drying it on filter Papa. Finally the grids were rotary shadowed at an angle of 7’ from an electron gun (Moor. 1970) with 1000 Hz carbon-platinum, as measured on the thin film monitor.
(h) Electron
microscopy
A Siemens Elmiskop 101 electron microscope operating at 100 kV accelerating volt,apcL was used with a 400 pm aperture in the second condenser lrns and a 50 pm aperture in the objective lens. Micrographs were taken at 27,000 x electron optical magnification as determined on a carbon grating replica from Balzers Union, Liechtenstein. The variation in rnagtlification for different specimens was 30/:, or lcsa (li. Portmann & .J. Hogo, rlnpul~lishctl rcsult,s). Micrographs wore recorded on Kodak c+lnrtror~ image plates tl~~~~clopt~~t :I( 20’ C’ f’or 4 min ill Kodak HRP dc:\-eloper (dillltiorr I : 4).
FIG. 2. Electron micrographs of Qb RliA-mplicase comploxaa. The preparations are described in Materials and Methods. Magnification: 110,000 x . A, 811 extended complex of Q/3 replicase and Q/J RNA. B and C, looped complexes of Qfi replicase and Qj3 RNA. Note the replicaso molecule at the base of the RNA loop.
Qb REPLICASE
BINDING
(i) Quantitative
SITES
ON
Q/3 RNA
371
analysis of the micrographCs
Selected molecules were printed at 10 x enlargement. Length measurements were et aE., 1974). The made with a Hewlett-Packard digitizer as described earlier (Portmann measuring accuracy was 0.75 mm, corresponding to O-003 Frn at the final magnification. The class sizes of the histograms wore 0.04 pm (Fig. 3) or around 0.016 pm (l%, Figs 4 and 6).
3. Results It has been shown earlier that incubation of Qp RNA with Qp replicase under conditions of RNA synthesis but’ in the absence of GTP and host factor I to preclude initiation, leads to formation of a stable complex which initiates RNA synthesis upon addition of the missing components (H. Weber, unpublished results). Such complexes first’ at low formaldehyde concentration at were fixed in a two-step procedure: 37”C, to stabilize the protein-RNA complex, then at higher formaldehyde concentration, at 63”C, to denature the RNA. Under the electron microscope the stained and shadowed preparations showed extended filaments. 60% of which were associated with a single blob. Among bhe strands with blobs two characteristic forms were
64
0 25
05
0.75
IO
Distance (pn) FIG. 3. Histograms of the location of Qp replicate binding sites on Q/3 RNA. The preparation of the complexes is described in Materials and Methods. Abscissa: distance of enzyme molecules from the RNA end. (a) 44 extended complexes of Qfl replicase and Q/3 RN.4. The end of the shorter RNA segment was positioned at the origin of the histogram. (b) 62 looped complexes of Qfi replicase and Q/3 RNA. For each complex, the lengths of both the longer and the shorter branches were plotted separately (differentially hatched areas). The black outline represents tho summation curve of the 2 individual distributions.
1172
Jf.
.I.
\'01,1,I~:Z\2~EII~E:li
KY'
.-I I,.
ol~sr~rved : ( I ) OIW third comprised fully extended filaments u:ith >\ single I)lol~ loc:atwl at around 300,, from one end of the molecule (Figs 2A and 5) two thirds \IW(’ strands with a loop and a blob located ;I,t the ba,se of t’hr> loop (Fig. %H and a(‘). Since in a11 wntrol exprrimrnt~s in \j.hich replicase was omitt,cd such I)lohs \\ VU~ tlxtremely rare and were not, observed in conjunction with a ~OJ) (in no castL among 350 filaments inspected). MY considered them to represent Q/3 wplicaw moleoult~s l)!)und to Qp R’NB. Accordingly. \ve will henceforth call thrw blobs wplicasc* tnolmllw.
The proportion of broken strands was variable in different experimctnts. possii)lJ due t*o differences between individual RNA batches as well as to variations in spreading. We t’hereforc selected the longest strands for the quantitative analysis (abow I + pm) a,nd considered these to be full length Q/3 RNA molecules. The longest strands with a bound replicase molecule \I.ere 1.7 pm. Qfl REA wit,hout replicaw. measured on (~xprriment,al or on control grids. showed strands with a length of up to 1.9 pm. Previous cbxperimcnts hare shown that after treatment of Q/3 RNA-Q/3 replicase complexes with T, RKase RNA fragments corresponding to about 350 nucleotides are retainc~tl by. C&3 replicase (Meyer. Weber & WFeissmann, unpublished results). It is possible. but difficult to ascertain, t)hat the apparent short,ening of t#he RNA by 0.2 pm (ahout 470 nucleotid&) is due to the coiling of RR’A ont’o thc& enzyme’. WV first analysed the complexes wit,11 110 RNA loop and mapped the positions of the rcplicase molrcules. In no case did we find a complex which had more t’han one wplicase nloleculc associated with the RPL’A. This \\.a~ also bhe (;ase \vhen \vt: increased the enzyme-t)o-RNA ratio I)y a factor of ten. compared to the standard procedure described in Materials and Methods, to give a molar ratio of about, 30 : 1. At a moln,r enzyme-t,o-RNA ratio of about 3:l some blobs not associat)ed with RSA were observed: the amount, of unbound blobs \vas substantially greater at a molar rat,io of 30 : 1, Figure 3(a) gives the histogram of the location of 44 replicaw molecules on t,hc R&A. The strands ww oriented such that the end of the shorter A rela,tivel>, hoad region wgment, was positioned at the origin of the histogram. of replicaw binding sites around 0.5 pm was obtained.
10 Qp RNA mop units p/o)
Fro. 4. Histogram of Q,9 replicesc binding sites on Q/3 RNA in looped complexrs. The preparat~ion of the complexes ia described in Mat,erials and Methock. Abscissa: position of binding sita in map units (the length of each RNA molecule was set to 100%). 62 complexes wvw analysrtl. The end of t,he shorter RNA branch was placed at the origin of t)he histogram.
374
H.
J.
VOLLENWEIDER
E!Z’
:iL.
The location of the replicase binding sites on the RNA of the looped complexes is shown in Figure 4. Again we positioned the end of the shorter RN,4 branch at the origin of the histogram. Since only 6% of the RNA strands without a bound replicast: molecule showed any kind of a loop (among 350 strands inspected) and since in Hit! looped complexes the replicase molecules were located always at the base of the loop, we conclude that the appearance of the loop was due to interaction of Q/3 replicaso with two sites on the RNA. The mean positions of these sites were at 0.46&OGl pm and at 1.03+0.07 pm from the end of the shorter RNA branch. In order t’o eliminate effects like variations in electron optical magnification or different extent of stretching of the RNA during spreading, we normalized the data, taking the length of each individual RNA molecule as 100%. The binding sites thus obtained \\ere located at. 29-5&2*5o/o (map units) and at 65f3.5% from one end. In Figure 3(b) we plotted the lengths of both branches of the looped molecules. The mean values for the two peaks in the histogram thus obtained were 0.46+0.04 pm (the same as described above) and 0.55kO.06 pm. No replicase associated with RNA could be observed under conditions which are specific for binding to only one of the two sites (Weber et al., 1974). As a control to these experiments \ve incubated E. coli RNB polymerase with Q/I RNA under our standard replicase binding conditions (Fig. 5A) as well as under conditions optimal for the binding of this enzyme to T7 DNS (Portmann et al., 1974) (Fig. 5B). Several (up to 5) enzyme molecules were bound to each RNA strand and their distribution along the Q/3 RNA strand was clearly random, except for some selective binding of enzyme molecules to termini (Fig. 6; orientation of the strands was again such that t#heend closest to a polymerase molecule was at the origin of the histogram). Only in 7% of the complexes was there a loop, of variable size, associated
Qp RNA map units P/d
FIG. 6. Distribution of DNA-dependent RNA polymer5se molecules bound to Q/l RNrl. The complexes were prepared as described in Materials end Methods. Abscissa: position of binding sites in map units (the length of each RNA molecule was set to 100%). The end of the RNA closest to a polymerase molecule was placed at the origin of the histogram. Blank colwnns: Black columns : binding coru s&me incubation conditions as for RNA-&B replicase complexes. ditions used by Portmann et al. (1974) for DNA-dependent RNA polymerese of h’. COG.
Q/3 REPLICASE
BINDING
SITES
ON
Q/3 RNA
375
with the bound enzyme. These findings are in agreement with the observation that when complexes of RNA polymerase and Q/3 [32P]RNA were treated with RNase T, the RRA fragments retained by the enzyme could not be resolved into discrete bands by polyacrylamide gel electrophoresis, which also suggested that binding did not, tske place at specific sites of the Q,4 RNA (data not shown).
4. Discussion We should emphasize that for technical reasons the data used for obtaining the hist,ograms could not be collected in a random fashion. Only those regions of the preparation could be analysed where the uranyl acetate st’aining was smooth and clean. and where the carbon-platinum shadowing gave a good signal-to-noise ratio. Furthermore, a variable number of RNil molecules was broken, making a rigorous selection of the longest strands necessary. Since the selection had to be done on several levels (staining, shadowing, association with enzyme, RNA length), it was impossible to estimate the yield of complexes which could be used for the mapping of replicase binding sites. The maximal length of uncomplexed Qp RNA reported in this work (1.9 pm) is clearly greater than has been described earlier (Koller rt aZ., 1971, 1.14kO.12 pm; Yuh Chi & Bassel, 1974, l*ll&O.ll pm; Sogo et al., 1974, 0.83kO.14 pm). This increased length of single-stranded nucleic acid molecules spread with the BAC method has already been observed with denatured T7 DNA (Vollenweider et al.. 1975). Under the assumption that unbroken Qfi RNA contains about 4500 nucleotides (Boedtker, 1971; Reijnder et al.? 1973; Yuh Chi & Bassel, 1974), we calculate for the spreading conditions of the present work an internucleotide distance of the order of 1 ,r(. It is not clear why Q/3 R1L’A complexed with Qp rnplicase appears to be shortened 1)~ about 0.2 pm. It is not possible to correlate the molecular weight of the replicase molecules with their apparent diameter since the latter depends on the amount of stain bound, which is undetermined and variable under the conditions used (Zobel $ Beer, 1961). The present work has shown that two thirds of the Q/l RNA-Q/I replicase complexes observed under the electron microscope contain an RNA loop with an enzyme molecule attached to its base. The loop constitutes about 3596 of the genome length, while the two free branches comprise about 30% and 350,x of the length, respectively. showed loops, and these Only 6$‘{, of the free RNA strands in the same preparations were of varying size and at diverse locations. Studies designed to reveal the secondary structure of Q/3 RNA (A. Jacobson & H. J. Vollenweider, unpublished data) shorn tha,t under less rigorous denaturing conditions a, small loop (about 0.1 pm in size) regularly occurs very close t’o one end, while loops as seen with the replicase-RNA complexes could not be found. We therefore believe that the loop in the complex comes about by the crosslinking of one Qp replicase molecule to two distinct sites of the RKA which are linearly far removed from each other, but, may of course be spatially adjacent in the native molecule. We have never observed RNA filaments to which two replicase molecules were bound, even when the molar ratio of enzyme-t,oRNX was increased tenfold to 30. This is in contrast to the control experiments with DNA-dependent RNA polymera,se, where several. np t)o five, molecules were bound t,o one Q/3 RNA. This finding is compatible with t,he assumption that Q/? replicase binds simultaneously or in rapid succession to both sites to form a stable looped complex, and that t,he extended complexes with one bound enzyme come about by
37Ci
r-I
.I
1.0 1, I, b: N \\’ E I I ) E I< E 7’
I 1.
parCal disruption of bhe looped complex during tixatiott or spreading. ‘1’11~ I1istjogratil of Figure 3(b) shows the length distribution of \wt,h 1)ranches of eaclt of the I00ped trtolccules. The tn.0 result,inp peaks overlap partially and if t,he suttttttation wrv~ is drawn (see Fig. 3(b)), one obt,ains a distrihubioti which resemhlrs the histogram fot the cxtendcd complexes (Fig. 3(a)). l’hcrefow. in t~lte extended c~m~plexes. tltc: r~plicase could 1)~ atItached to one or the, ot,hw site with al)out ~~(luitl frqu~~nv~~. Tlte observation. that under conditions where biochemical experitttettts Itav(B demonstrated stable binding to only one site no complexes are found. tnight also he explained by disruption of the complexes during the preparation. ‘I’he electron microscopic ohservat,ions are compatible witlt our previous I)iochemical findings. It \\‘as shown earlier t)hat if [“‘TJQ@ RNA-Q/3 replicase complrxcs, formed under the same conditions employed in the present work. wew treated with RNase T,. several RNA segments remained hound t,o t’hr enzyme. One 1at.g~ RNA piece (fragment S-3. 100 nucleot~ides) was sho~vtt to be derived from the region preceding the coat, cistron. i.e. ahout 31”,, of the genotne length from the 5’ end (Weher of al., 1971). while three othtbr fragments (M-2. M-5. and M-I 1. \rith 164. 60. and 21 nucleotides. respectively) probiibly originat,ed from a region Lvit(hin the twginning of t)he replicase cistron. about 47 t,o A()“,, of t#he gcnomc tcnpth from the 5’ end (Weber rt rrl.. 1974; Meyer rf 111.. 1975). The position of t)he S site. which could he located fairly accurately on the genomc (cf. Fig. 1). is pract,ic:ally identical wit,h that of one of the binding sites identified by elect,ron microscopy. The position of t,hr other site, as det’ermined I)y electron tnicroscopy, falls just outside the region from which the M fragments are believed to originate. Since the biochr:mical mapping of the M site was not ver) ibccuratt~ we believe t’hat our data are contpat,iItle \qith the t\vo positions being identical. Alt~crnat.ivcly, replicasisslnann. C.. Billeter, M. A., (inodman. H. M., Hindley. ,J, & Weber, H. (1973). *-Jr~lol. ICer.
Hiochem.
42,
303-328.
Yuh (‘lli. Y. & Bassel, A. R. (19i4). J. Viral. 13, 1194 1199. %oi)cal. (‘. Ii. h Hfbrr, M. (1961). J. Riophys. Hiochem. Q/to/. 10. 333
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