The in vitro transcription units of bacteriophage φX174

J. Md.

Biol.

(1976)

103, 711-736

The in vitro Transcription III.

Initiation

Units of Bacteriophage

with Specific 5’ End Oligonucleotides 1,. H.

C’nlifornia

AND

R.

L.

30 June

of in vitro $X174 RNA

sTWSHEIMER

L)ivision of Biolofgy of Technology, Pusadetu.

Institute

(Received

SMITH

#X174

Culif. !Il188,

1975. and irv, reaised fom

C:.S’.d.

15 I~ecevvder 1975)

Four major 5’ end oligonucleotide sequeucrs are detected in 5’ proximal]\ labeled in ,uitro bacteriophage +X174 transcripts. These are correlated to the three major initiation sites previously identified by preparative hybridization aud nucleotide fingerprint analysis. In addition, the 5’ end oligonucleotides of tile several discrete components isolated by preparative slab gel electrophoresis, also 5’ proximally labeled, are identified. The results confirm the genetic locations of tlw tllretb major transcriptive initiation sit)es prrviously postulated.

1. Introduction lkherichia

coli RNA polymerase holoenzyme can recognize and respond to specitic and termination sequences in vitro with template DNAs from a number of sources. Among the genomes for which the in eitro transcriptive units have heen highly characterized, both with respect t,o their genetic or physical location and their 5’ proximal nuoleotide sequence, are bacteriophage fd (Sugimoto et al., 1975; Seeburg & Schaller. 1975), bacteriophage T7 (Dunn & Studier, 1973a,h; Minkely & Pribnow. 1973: Rosenberg et ul., 1974), E. coli lac (Maizels, 1973), and bacteriophage h (Lebowitz pt al., 1971 ; Blattner & Dahlberg, 1972; Dahlberg & Blattner, 1973). Examples from these data demonstrate that, in vitro, RNA polymrrase holoenzyme alone can synthesize transcriptive units identical to those formed hy the in zjivo expression of a particular genome. We have previously described the isolation and sequence determination of four major kinds of 5’ triphosphorylated oligonucleotide from iv6 vitro bacteriophage +X RNA (Smith et al., 1974; Grohmann et al., 1975). In the two preceding papers, we have demonstrated the existence of several discrebely sized units of in vitro (6X174 t,ranscription (Smith & Sinsheimer, 1976a) and have determined their location rela,tivc to the $X174 genet’ic cleavage map (Smith & Sinsheimer. 19763). Here we report, t’he initiation

correlation regions

of the on the

specific

5’ end

oligonucleotides

with

each

of the

specific

initiat,ion

4X 174 genome.

2. Materials (a) ,‘!P-labeled

and Methods in vitro

4-Y RNAs

(y-“2P)-labeled ribonucleoside triphosphates (obtained from ICN at spec. act. of 76 Ci/ mmol (ATP, GTP) or 35 Ci/mmol (CTP, UTP)) are lltilized in the synthesis of (Y-~~P)labeled in vitro +X RNAs as described by Smith et al. (1974). ( E-~~P) proximally labeled in vitro $X RNAs are prepared and isolated as described by Smith 85 Sinsheimer (1976a). 511

i 12

I,.

H. (b)

SJlII’I’H

AS

I) li.

Oligonucleotirle

1,.

SLNSHEIMEK

jingerprint

analpin

Fingerprint analysis is as described by Barrel1 (1971) with minor mc)clific.atiotis (Sttiit II et aZ., 1974). 32P-labeled RNA for fingerprint analvsis is prccipitatr~tl vvitli etllanol (iri polyallomer tubas) with 16 to 20 pg ca,rricbr E. coli soluhlo RNA. Tlic pelleted material is washed once w-it,11 5 ml cold 70:/, ethanol, repelleted, dried h&fly i~iitler rc~duced prossurc), and redissolved in -100 ~1 deionized water. The RNA is then evaporated to clryness in a siliconized glass tube, and redissolvad in 2 to 2.5 ~1 0.01 nr-Tris.HCl (pH T.!)), 0.005 ~RIEDTA containing an appropriato amount of either ribonuclease T, (Calbiochrm) OI ribonnclease A (Sigma, grade IDA). Enzymo t,o subst,rato rat,ios of 1 : 10 (\v/vv) are employed and digestion is at 37°C for 40 min. The dig&s are t,hon applied to cc~ll~~losc~ acctutcb strips Tl it, second-dimension llornochroillatofor first-dimension ionophoresis (Barrell, 1971). graphy (DEAE-cellulose, thin-layer) is developed with a mixture of 3% 30 min.hydrolyzed (3 parts) and 20 min-hydrolyzed (1 part) homomixt,ura C (Barroll, 1971). After l~ornochromatography, plates are autoradiographed (Kodak medical X-ray film RP-54). Spots are cut out of the plastic-backed thin-layer plate and counted in tolunne/fluor count,ing fluid for quantitation of radioactivity. The approximate chain lengths of all of t,he oligonucleotides on the chromatogram arc estimated from tlio posit’ions on the plate of the spots of known sequence. The radioactivity is then expressed as cts/min per nucleotide or relative molar yield. Relative molar yields for t,he oligonucleotides within a givcii fingerprint are obtained in the following way. An oligonuclcotide present in one copy per transcript (i.e. a 5’ end oligonucleotide) is identified. The cts/min por nucleotide for cacli oligonucleotide in a given fingerprint are divided by the cts/min per nucleot’ido of tlicr oligonucleotide judged to be present in a relative yield of I .O. For nucleotide composition analysis, t,tlo spots are removed from tolueno, washed extensively with absolute ethanol, dried, scraped into a suction tieviccl, and elut,ed with 300 to 400 ~1 0.3 ar-NH,OH. The elnted radioactive material (SOq/, of the ct,s/min on tlie original spot) is ovaporated to dryness and redissolved in 10 ~1 0.01 nr-EDTA. A mixture of ribonucleases (Smith et al., 1974) is addod for digestion to rnorrorulcleotides. Digests are fractionated by ionophoresis at, pH 3.5 on Whatman DE81 papor (for resolut)ion of pppXp compounds) or Whatman 540 paper (for resolution of Xp). (c) Preparative

nitrocellulose

jilter

hybridization

Preparative hybridization of (E-~~P) proximally labeled in vitro +X RNA to endonuclease fragment filters is performed in the following way. Nitrocelhrlosc filters (13 mm diameter, HAWP, Millipore) are used to bind 10 pmol of a particular alkali-denatured endonuclease +X replicative form I DNA fragment (see Smith & Sinshcimer (19765) for details). Filters are washed with 10 ml 6 x SSC (SSC is 0.15 nr-NaCl, 0.015 w-sodium citrate) three times, blotted, air dried and vacuum baked for 2 11 at 80°C. Hybridization is as before (Smith & Sinsheimer, 1976b) in 65 “/ formamide, 2 i( SSC and 0.1% sodium dodecyl sulfate for 72 h at 25°C. After hybridization, the washed filter (5 times, 5 ml 2 x SSC) is treated with ribonuclease A (20 pg/ml, heated, Sigma grade IIIA) or T, (2 units/ml) in 1 ml 2 x SSC, incubated for 40 min at 22”C, washed a,gain (5 times, 5 ml 2 x SSC), and blotted dry. The filter is t,hen treated with 1 ml 0.15 &r-sodium iodoacetate, 0.1 M-sodium acetate in 2 x SSC at 54°C for 40 min. as described by Zain et al. (1973). dry and the RNA eluted by The filter is washed again (5 times, 5 ml 2 >: SSC), blotted 2 h incubation at 45°C in 2 ml 7074 formamide in water (Hayashi $ Hayashi, 1972). Tho cluted RNA is precipitated with ethanol, washed with 70g0 c+hanol, roprecipitated and prepared for fingerprinting as described abo\-cl.

3. Results (a)

5’ oligonucleotides

labeled by (A-““P)-labeled

nucleoside

triphosphates

Figure 1 shows autoradiograms of the ribonuclease T,[y-32P]BTP (Fig. l(a)), T, [y-32P]CTP (Fig. l(b)) and RNAase A[y-32P]GTP-labeled (Fig. 1 (c)) oligonucleotides present in +X RFI-directed in vitro RNA. The sequences shown are those determined

5’ END

SEQUENCES

OF IiV

FIG. 4;

l(a)

PIITIlO

c$X HSAs

7 13

714

I,.

H.

SRII’L’H

.\Nl)

II.

L.

SlNSHI’:lJlEH

FIG.

I(c)

fingerprints. First-tlimension ionophoresis is from Frc:. 1. Autoratliograms of I-dimensional left to right ; seconti-dimension homochromatography from bdtom to top. Dotted circle marks position of yellow dye marker. Sequences are from Smith et al. (1974) and Grohmann et ul. (1975). (a) T, RNAase digest of [y-32P]ATt’-labeletl irt dro c$S RNA. (RNA prepared wit,h 3.5 mont,hold RNA polymerase.) (b) T, RNAase digest of [y-32P]~‘T1’-labele~ in P&O @ RNA. (RX.4 prepared with 3.5 mont,hold RNA polymerese.) (c) RNAase A digest of [y-32P]GTZ’-labeled irl vitro +-‘i ItNr\. (RN/\ prepared with 0.5 mont,h01~1 RNA polymerase.) S~~quencns in parent,hr.ws are ttssumctl. ~q~~d:pPupPyp is OIIVof thr f~~ll~~w~ng seqwnw~: ppp(:pGpUp. pppC+A~K!p, pppBpGp(‘p.

il6

I,.

H.

SMITH

.ANl)

It.

L.

SISSHEIMEK

previously by analysis of the (r~-“~P)-lal)rl(~d 5’ cbntl c~lipol~llclrot,i(lf~~ (Smit,h VI rrl.. I974 : Grohmann PI NI., 1975) ; sc~qucnc~es in l)arc‘nt hescas WI’~’ a.ssumr~d. 1y-““PIITTP does not label any part,icular oligonucleotides: t,he small amount of [ Y-“~P]UTPlabeled, T,-digested RNA is widely spread throughoub many weak spots (data not shown). For labeling conditions see Smith et al. (1974). Table 1 lists these (Y-~“P)labeled oligonucleotide sequences and t’heir relative molar yields. The a,gc of the RNA polymerase preparation used affects t,he relative molar yields of the minor oligonucleotides in comparison to the major ones. The data in Table 1 refer t,o RKA prepared with three to four month-old RNA4 polymerase holoenzymc~: here the major

oligonucleotides (pppApUpCpGp

-t

pppxp (4p)2.sUpCpKJp)2 Gp -: pppGpApUp i-

pppCpGp) account for 50 to 600/(,, of the total (Y-~~P) incorporation. can be higher (up to SO’$‘{,) in RNA prepartd with RNA polymcrasr one month old. TABLE

Relative -,

rnohr

1

yields of (y-““P)-labeled Sequence

PPP*PUPCPGP(C) PPPAP(AP)JJPCP(UP),GP(G) PPPAP(AP)~UPCP(UP)~GP(G) PPPAPUPGP(G) 1 other pppAp- oligonacloot.icles

This proportion t,hat is less than

oligonucleotidw Relative

molar

yield

I.000 0.258 0.510

PPPGPAPUP(G)

0.488 0.549 0.250

(PPPGPUP) (PPPGPCP) kwJW’W’yp) t 1 other pppGp- oligonr~clo~,t,i(l~~ PPPCPGP(A) PPPCPCPGP 1 pppUp- oligonucleotides

0.148 0, 123 0.141 0,046 0.560 0,265 0.061

Relative molar yields are normalized to pppApUpCpGp = 1.0. Original yieldd ata, is in terms of pmol (y-3zP)-laheled oligonucleotide per pg product in vitro RNA, where the conditions of synthesis of [y-32P]ATP-, [y-32P]CTPand [y-3ZP]GTP-labeled in vitro RNAs were identical (see Smith et al., 1974). t pppGpPupPyp is one of the following sequences: pppGpGpUp, pppGpApCp, pppGpGpCp.

For example, the 5’ end oligonucleotide pppApUpGp in Figure 1 is present in a yield of 0.488 relative to pppApUpCpGp (relative yield 1.00); this fingerprint is of RNA prepared with three to four month-old RNA polymerase. With RNA polymerase that is less than two weeks old, the yield of pppApUpG relative to pppApUpCpGp is less than 0.1. In the T, fingerprints of 5’ proximally labeled RNA (see below). the age of the RNA polymerase used is one to two months, consequently the yield of pppApUpG (again relative to pppApUpCpG) is slightly increased, 0.1 to 0.2. The relative yields among the major oligonucleotides, however. are essentially unchanged with the age of the enzyme. It is of course conceivable t’hat some of these “minor” in vitro initiation sequences are actually significant starting sequences in viva. This, however, does not appear to be the case, at least for the pancreatic RNAasr oligonucleotides (see Discussion).

5’

ENI)

SEQUEN(‘ES

01’

IAl’

1’1’l’RO

(II) 5’ end oligonucleotide sequences present labeled in vitro $X KNA

$S

I;N.\s

71;

in. 5’ proximally

Figure 2 shows autoradiograms and schematic representations of homochromatograms of RNAase A((a) and (b)) or T,((c) and (d)) digested, 5’ proximally labeled (30 s. 25”C, 50 PM-XTP, see Smith BE Sinsheimer, 1976a), “GBO-chased,” in vitro 4X RNA. The four major kinds of 5’ end nucleotide sequence detected by (y-32P)labeling are also synthesized during the 5’ proximal label pulse. The sequences of the other numbered oligonucleotides have not been determined, therefore they will subsequently be referred to by number. Unnumbered spots are never present in relative molar yields above O.Fi. (v) RNAase

A 5’ end oligonucleotides present it& hybrids replicatiue form DNA fragment filters

to endonzdease

RNA equivalent to that fingerprinted in Figure 2 is hybridized to various endonuclease +X replicative form (RF)1 DNA fragment nitrocelluloae filters, as described in Materials and Methods. Filters for RNAase A fingerprinting are RNAase A-treated after hybridization to remove single-stranded sequences. This RNAase treatment (20 pg/ml, 2 x SSC, 40 min, 22V) is the same used for the analytical filter hybridizations described in the preceding paper (Smith & Sinsheimer, 1976b), but is more severe than treatments sometimes used (< 1 pg/ml, 2 x SSC, 40 min, 22°C) for removing single-stranded RNA from preparative hybrid filters (e.g. se? Zain et al., 1973). While the RNA subsequently eluted from filters treated in this way is somewhat fragmented (our unpublished observations), it is likely to contain very little noncomplementary sequences. Figure 3 shows autoradiograms of RNAase A fingcrprints of 5’ proximally labeled RNA hybridized to Hin 4 (a), Hin 8 (b) and Hpa 3 (c) fragment filters, and treated as described above (see Smith & Sinsheimer, 19760, for definition of Hin 4 etc.). Comparison with Figure 2 reveals that the pppAp(Ap),-, Up(C) oligonucleotides are specifically complementary to sequences in Hin 4. Similarl) Hin 8 is the origin of the pppApUp dinucleotide. (A small yield (see Table 2) of pppdpUp is seen in the Hin 4 fingerprint. This may be due to other minor pppApU1) starting sequences (e.g. pppApUpGp) within Hiu 4 or perhaps derives from the pppAp(Ap)n Up(C) sequence, where n = 0. The oligonucleotide pppApUpGp is found in the T, fingerprint from the Hin 4-hybridized RNA, but’ also in the T, fingerprints from the other hybrid regions. It is not clear if Hin 4 is the only region from which pppApUpGp originates.) Hpa 3 specifically hybridizes the pppCp and pppGpApCp oligonucleot,ides. Table 2 lists the relative molar yields of all of the RNAastb A oligonucleotides found on these plates, as well as the spots from the unhybridized RNAase A fingerprint in Figure 2(a). In each case the relative molar yield of t,he 5’ end oligonucleotide(s) found to be specific for the fingerprint is adjusted t,o 1.0. FOI the Hilt 4 fingerprint, the yield of ppAp(Ap),Up + pppAp(Ap),Up is taken as 1.0. as is the yield of pppCp -t pppGpApUp for the Hpz 3 fingerprint. Using the chain lengths and the molar yield data, a number for the total length (in nucleotides) ot the 5’ proximally labeled region of 80 to 100 nucleotides can be calculated. This is in reasonable agreement with the size range of the partial transcript lengths measured for these 5’ proximal pulse label conditions (Smith & Sinsheimer. 1976a). The dat)a do not eliminate the possibility that the pppCp and pppGpApVp oligonucleotidrz originate from independent init,iations in different parts of the HP:& 3 region. How~vt~r

FIG. 2(a)

5' END

SEQUENCES

OF IA

I-ZI'RO

,#S RNAs

RNase A

0 I

0’” 0 0

20

519

RNase TI

(d)

E

-n6

0

20

0

21

25

0

22

FIG.

:3(c)

FIG;. 3. Autoradiograms of RNAase A fingerprints of 5’ proximally I~ht:letl RNA hyhrithzed to endonuclease $X RF1 DNA fragment. nitrocrlldoso filters. S~qucnccv shown aw fhow (letermined by Smith et al. (1974). (a) RNAase A digest of the Hire 4 hybrid RNA; (b) RNAwP A (ligwt. 01 the Hin X hybrid RNA : (c) RNAaw A (1igt.d IIf thr Hpu 3 hybrill RX;!.

5’

END

SEQUENCES

OF

19

‘l’ABI,E

RNAase Oligonucleotide

1 ., 3 4 5 PPP(!P(G) 0 7 x 9 10 11 PPP.~PUP(C) 14 13 14

15 pppGpApUp(G) 16 17 18 PPP~P(~P)zUP(~) PPP~P(AP)~UP(C) 1 !) “0 t Oligollucleotitle(s)

Hin

4

14.x 6.1 11.2 0.8 5.3 .‘().I 0.1 0.1 ‘ 0.1 2.0 4.9 4.6 14t

“: 0.1 0.8 0.1 sr0.1 CO.1 0.0

1.5

0.5

0.1 CO.1 0.2 0.2 0.7 0.q oq 1.8

1.5

0.4

molar

yioltl

d&a

T, 5’ end oligonucleotides

3

84 2.3 2.X 3.5 2.1 Wtjt 2. 1 0. 1 3.X O.9

2.3 0.1 ( 0.1 4.4 0.5

2.3 6.1 0.4t i 0.1

4.5

i 0.1

0, 1 -: 0. 1 ;; 0. 1 0.x (, 0.1

0.1 io.1 ...().I 0.1 2.4

(soo Matcrials

the sequence of the T, oligonucleotide pppCpGp(A) their origin could conceivably he a common initiation (d) RNAase

Hpa

hybrid

0.1 0.8

3.5 0.0 3.0 0.5 w1t 0.w

8

0.4

2.0

usotl to normalize

Hin

hybritl

18.9 2.1 2.6 5.5 3.9 CO.1 0.1 0.7 0.7 1,L’ I.!) 2.X

0.4

725

RN.-\s

relative molar yields

hybrid

304) 4.2 IS.0 8.X 3.5 1 .5 2. I 1.5 4.2 2.9 4. I I.1 2.5 3.2 1.1

$S

2

A oligonucleotides;

Unhybridizd

I’ITRO

overlaps point.

from hybridized

ant1 Methods,

swtion

pppGpApUp(G),

(b)).

so

[“‘PIRNA

The filters for RNAase T, fingerprinting are T,-treated prior to elution of the RNA. This treatment is considerably milder (2 units/ml equals about 1 pg RNAaxtr TJml) than the RNAase A filt’er treatment conditions, and consequently larger amounts of non-complementary sequences contaminate the final preparations. Figure 4 shows autoradiograms of the T, fingerprints of 5’ proximally labeled RNA. preparatively hybridized to Hin 4 (a), Hae 2 (b), Hin 8 (c), Hue 3 (d), and Hpa 3 (e). Inspection of these Figures and the data in Table 3 generally confirm the data of the RKAase A fingerprints (Fig. 3, Table 2) as to the origin of the major 5’ end oligonucleotides. Hin 4 and Hae 2 hybridize identical sequences within the 5’ proximally labeled RNA. The pppAp(A),.3UpCp(Up),Gp(G) sequence is therefore associated with the gene H-gene A initiation region. Proof that these two sequences arise from ambiguous initiation at the same promoter rather than from different sites within Hin 4 awaits the elucidation of further 3’ nucleotide sequence information. The Hk 8 fragment hybrid contains the pppApUpCpGp(C) end sequence, which must therefore he associated with the gene A-gene B initiation site. Hpa 3 selectively hybridizes both pppCpGp(S) and pppGp. also in agreement with the fingerprints of Figure 2.

i:rc:. 4(h)

I.‘Ic:. -l(c)

FIG.

1((l)

Oligonucleotido

Unhy bridizctl

Hia 4 hybrid

Htre 2 hybrid

Hire X hybrid

3.4 0.5

3.1’ 0.X

9.i 0.5

1.4 3.0

0.5

3.4

t

0.4 1.7 24, 3.5 I.4 0.G 0.5 0.x 1.7 0.3 Wti 0.3 I.1 0.3 0.5 0.X 1,ot 0.8 0.3 Il.4 0.i O,(i I).!)

0.i 0.3 0.7 0.2 0.5

3.2


CO.1
Relative molar yields determined as described in Maturialx t Oligonucleotide(s) used to normalize molar yield d&t:%.

63

ro.1 1.1 0.3 4.1 3.9 0.x 0.1 0.X ‘.O.l 1.7 -, 0.1 . . WI II.1 I .(i

.-. 0.1 ‘._ 0.1 ‘. 0.1 1 .ot .: 0.1 0.4

co.1 .I).1 -:w1 “CO.1 ..,CJ.I 0.1 0.1 -. 0.1 co.1

Hne 3 hybrid

Hpn 3 hybrid

2.8 0.G 1.1 3.0 0.3t 0.G

1.1 0.1 0.1 I.8 0.3-F 0.2 I.7 0.1 -CO.1 1.X o.it <‘().I 10.1
2.4 1.2 0.3 2.3 0.7t 0.7 0.8 0.G 0.5

1 ,3 0.2 0.9 0.3 1.0 1.1 CO.1 0.2 0.5 0.8 1.2 0.1 0.0 0.2 0.3 1.5 0.2 0.3

I .4 0.8

-co.1 1.2 1.3 .
1.0 1.4 ‘.. 0.1 i 0.1 -. 0.1 0.4 1.5
and Methods.

The Hae 3 hybrid fingerprint (Kg. 4(d)) indicates that most if not all of the Hpa 3 hybridizable oligonucleotides are also hybridizable to Hae 3. However, several (if not all) Hin 8-specific oligonucleotides, notably pppApUpCpGp, are missing from the Hae 3 pattern. Therefore, the actual gene A-gene R initiabion site must be somewhat, t,o the 5’ side of Hae 3 (viral strand), but within Hin 8. A T, fingerprint of 5’ proximal label hybridizable to Hae 1 (data not shown) shows in no prominent Hae 1 -specific oligonucleotides ; instead most of the radioactivity the fingerprint is present as low yields of spots found in higher yields in the other (Hin 4, Hin 8, Hae 2, Hae 3, Hpa 3) hybrid fingerprints, presumably present due to the mild T, treatment employed, which did not, completely remove non-complementary sequences.

732

I.. (0)

HNA

aw

H.

T, ,fitrgvrpriMs

SMITH

.\SI)

l<.

f!/ iwlrrl~rl

1,. SINSHk:I1lEI{

r.-d’/’

,i’ j~ro.ritrwll,tj

IUIIP~PI~ CY~~~~~N~~W~~/.Y

Table 4 shows t,hc relativtb molar yklds of oligorluc:l(~otitlt,s prwc~~t in ‘I’, tingw prints of specific components of 5’ proximally labeled G50-chased RSd isolat~~ti from 20/,, acrylamide. 03qi, agarose slat) gels (Smith & Sinsheimw. I976a). Tlr~b autoradiograms of these fingerprinb art’ not, shown. since they closely resemhk t,lw autoradiograms of the T, fingerprints already presented. Table 4 contains th rdatiw molar yields for oligonucleotides present in these fingerprints: t h(* oligonucl~,otitl~, numbers refer to the same spots numhercd in Fipuw 2 (T, oligonucleotidrs). Component B. shown by analytical hybridization to contain squencrs from all

4

TABLE

RNAase

T, oligonucleotides relative

of 5’ prozimall,y molar

labeled

gd

hands:

yields Htlntl

Oligonucleotide

( ‘:3

(‘I 3.3 0.5 2.8

PPPGP

PPPCPGP(A)

PPPAPUPCPGP(C)

5

10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25 PPPAP(AP)JJPWUP),GP(G) 26 27 28 29

4.1 : 0.5 2.3 2.X 4.9 --9 .> 0.7 0.6 0.8 2.4 0.4 0.8 0.4 I.7 04 0.8 0.9 14t 1.1 0.4 0.7 I.1 1 ,o 1 .o 0.‘) 0.5 0.9 l-l.2 04;

1.x 0.3 0.6 2.4 t 0.1 0.6 0.X I.0 0.4 0.1 0.2 0. I I4 0.1 I),1 0.4 0.4 0.4 II.1 0.4 0.3 0.3 0. I 0. 1 0.2 0.3 I.2 Iq 0.2 0.2 0.1 0.6

Relative molar yields determined as described in Meterialn and t Oligonucleotide used to normalize molar yield data. $ Ran off in first dimension. 3 Assumed to be 0.3 (see Table 3) so t,hat, pppGp + pppCpCp hybrid).

2.1 0.2 I .*5 1.4 1 0.2 0.7 1 .6 4.6 0.7 0.1 0.5 0.2 I.8 0.1 0.1 0.2 1.1 II.2 0.2 0.3 1.ot 1.2 0. I II.2 0.2 1.0 0.4 I).2 0.1 0.2 0.1 0.1

2.1 0.2 0.i 2.5 s 0.2 1.X 1 ,o I .6 2.0 o,7t (I.2 0.8 0.x 0.6 1.0 0.2 I.1 0.1 I).!) 1.1 0.3 0.4 0.6 0.x I .:I (I.3 0.3 II.3 0.5 I.4 0. I II.2

Methotls.

1 .O (SW Hpo

3 or HOP :I

5’

ENI)

SEQ1’EN(‘ES

01,’

I,\‘

l~/‘I’/:O

4X

l:S.\>

7x1

essentialI> thrt~t~ initiation regions (Smith & Sinshrimc~r, 197(K). has a T, fingerprint identical to that shown in Figure 2(c) for total 5’ proximally labeled RNA. ‘l’ablc -I shows a similar quantitative pattern between the H and unfractionated fingerprint,s (see Table 3 for T, total 5’ proximal label fingerprint data). This is consistent with tlte finding that, region B actually contains three unresolved bands (Smith & Sinsheimcr, 1976~). Hand Cl. associated by analytical hybridization \vith the gene H-gent) d initiat,ion region, contains unit yields of only the pppAp(Ap)2.3UpCp(Up),Gp(G) sequences. Band C2 contains the pppApUpCpGp(C) sequence in highest molar yield. thus it’s assigned origin from analytical hybridization (the gene A-gene B region) is t*onfirmed. Similarly, since band C3 contains pppCpGp(A) in high yield (pppGp ran off the Figure in the first dimension), its assignment to the gene (T-gene L) initiator hi. analytical hybridization appears valid. The T, fingerprints for bands DI and D2 (data not shown) are essentially the satne as for bands Cl and C2, respectively. The daba of Tables 3 and 4 can be summarized as f’ollows. (1) The sequences present in t)and Cl are present in both t,he Hin 4 and Hue 2 hybrids. (2) The oligonucleotides of band (‘3 are present in both the Hpa 3 and Hue 3 hybrids. (3) Two large oligonucleotides present in band C2 (spots 20 and 24) are missing from bot,h the Hin, 8 and Hap 3 hybrids. ‘I’hereforc~. there may be it gap between Hi// 8 and Flae 3. instra.d of an orrrlap. . as much of band C:! If this \vtxrt’ thtb case. Hin, 5. adjacent) to Hi/l 8. qhoutd ttvl)ritlize as Hnr 3 does. Since this is not, the case (see Smith & Sinsheimer. 1976b). the possiI)ilitx arises that another Hin fragment. previously not identitied. may hc present I)rtwt:txn Hin 8 and Olin 5. This new fragment would have to be large enough to COVCI t,ht portion of the proximally labeled C2 tra,nscript, conbaining the T, oligonuc1eotidt.s 20 and 24 and the portion of C% lvhich hybridizes Mrrr 3 (e.g. spots 3 and 7). A fragtnmt of 20 to 30 nucteotides would do this, and would hc small through to hare rscap~tl previous detection (A. Lee. personal communication). IItrr 10 should also contain sequences complemtantary to the 5’ proximal region of band C2. However, the analytical hybridization data for proximally labeled hand C2 with the Hw fragment set (Smith & Sinsheimrar. 1976h) show that Hae 10 ttyhridizes very little if an,v C2. Ho\vcver, Hae 10 is the poorest sullstratr for nitrocellulost~ tiltcsr hybridizat,ion among all of the fragment)s : a vtary small amount of 4X [ 3H ]D?4r\ which is present as a hybrid detection standard, can bind the HUP IO filter (see wig. 1. Smitll $ Hinsheimer. 1976b). It is conceivable then that the 5’ proximal label of I)atid (‘2 does cont,ain sequenctbs complentrntary t 0 t hc Ifln~ 10 region but tttat sucll hybrids arts retained very poorly if at all to ttrt> Ror 10 fittttrs. and tht> “H ttortn;llizntiotr ~~~~o~~~tlur~~fails to csorrrct. for thib toss.

4. Discussion Hayashi & Hayashi (1972) have shown tltat alkalint~ di@s of i/e cir~o 4X17-I messenger RNA contain pppAp, pppGp, and a number of minor unidentified cornponents in addition to nucleoside monophosphates. Recentl,y, methods for t’ht> isolation of 5’ triphosphorylated oligonucleotides havc~ been developed (Soave it [cl., 1973 : Grohmsnn et al., 1975). We have employed these met’hods to isolate and identi@ the 5’ triphosphorylated nucleotides present in it/. &JO +X174 mRNA (A. Szalay. K. Grohmann, L. H. Smith & R. L. Sinsheimer, unpublished data). We find that’ the in &VI $X mRNA 5’ end oligonucleotides have mobilities and nucleotide compositions tlnit,ts similar to the in ~ilro oligonuclcotidrs pppAplTp. pppAp(Ap),.,Up. ant1

i34

I,.

H.

ANI)

SMITH

I<.

I,.

SlSSHEIRiEli

pppGpApUp. No pppCp is prcscnt in the i/l. viva $S inRNA. 111 t,his WS~NY~, tflk(* finding of ,Jorgrtlsrn d d. (IWI) t,hRt, E. CO& r;ll)irlly I;l~wI(~tl RNA (*ottt,;litts ottI>. pppAp and pppGp arc similar. Basically, thcbstt data indicat(~ that in ~w’tro KNA polymerase initiates chains predominant’ly with 5’ end scqucnccs t,hat ;we also found in the in vivo $X mRNA. The synthesis of pppCp containing oligonucleotides in vitro may be attributable to the ambiguous selection of the primary complementary nucleotide during initiation at the gene C-gene D initiation region. Maitra et al. (1967) found, for a number of templates, very little (5 to 100;: of total) initiation beginning with [y-32P]CTP: however, CTP initiations were shown to be five to ten times more frequent than UTP initiations. It may be that CTP-initiated transcription is of variable significance depending on the particular template or promoter. The fact that pppCp is not detected in vivo suggests that certain constraints on initiation selectivity which operate on RNA polymerase in the cell are removed when the enzyme is purified. 4X RF-directed pppCpGp synthesis is not a function of the age of the RNA polymerase, as is the case for many of the minor 5’ end oligonucleotides. Several independently isolated RNA polymerase preparations produced pppCpGp, regardless of age. The increased Base pairs

Cl R3

Hin t

Hoe I

RB PI

Hpa ---

5’Gene

I 1000

500

Z6,Z6b

I

I 2000

R7b

P4,

R6b

R6,

P4b 27 I-,

25

, -5 ,C, -D , am 16 am IO

I 3000

I 2500

R7,

P3 23

! ! ! A

----

R5

Zgi!IO

!

I 1500

I 3500 RI

P7

P2 p++-+!c-pI

z0 I

E

I 4000 RIO R9j R2

24

P6

I 4500

I 5000 R6,

I

R4

I

P5

ZI

22 I

,J,

--_ F

,

G

,

H Bands

,A Cl, DI

1 ~‘PPPAPMP)

3 i-S

‘JPCP(UP)~GP(G) Bands

,

_-_

0

CZ,D2

I ~‘PPPAPUPCPGP(C

) 0 Band

C3 1 ~‘PPPGPAPUP(G) PPPCPGP ( A)

0

w Terminator region

Gene ----

Base pairs

FIG. taken to right tion is et al.,

t 0

A I 500

, -B am 16

,C,

I 1000

I 1500

-D, am IO I 2000

E

F

,J, I 2500

6. Locations of the 5’ end oligonucleotide from Lee & Sinsheimer (1974). Initiation is 6’ to 3’ for the 4X viral DNA strand left to right. $X translation (and therefore 1972).

I 3000

, G I 3500

,

I 4000

H

,

I 4500

I 5000

A --I

initiations. Composite genetic-cleavage map is points are marked by vertical arrows ( 4 ). Left (same sequence as in vi&o RNA), and t,ransoriptranscription) in wiwo is also left to right (Benbow

5’

END

SEQUENCES

OF

IX

I’ZTRO

+S

RNAs

i35

synthesis of minor 5’ end oligonucleotides with ageing of the RNA polymerase is probably a result of sigma subunit inactivation (Sugiura et al., 1970). In Figure 5 the 5’ end oligonucleotide sequences for each initiation region have been added to the previously mapped units of irk vitro $X t,ranscription. It would be uf great) interest to know if t’his picture is also correct for thr control of 4X tramvription

during

infections

of’ El. coli.

\\‘cr arcs q-atoful to Dr J. u’. St&t ‘I’llis wstwrch v as s1qq1ort,~tl ill part Hrv~,ltll.

for llis clemoustration 1~) r grant GM1 3.554

of tht? sequencing frown tilt! National

procedures. Institutes

of

IZEFER,ENCES ~~EL~JY~H, B. U. (1971). Proc. Sdeic Acids Res. 2, 751-779. Benbow, R. M., Mayo& R. F., Picclli, J. A. & Sinsbeimer, H. L. (1972). J. Viral. 10, !)!I114. Bluttner, F. R. & Dahlberg, J. E. (1972). X&we Xecu Biol. 237, 227F232. Dahlberg, J. E. & Blattner, F. R. (1973). In virz~s Research (Fox, C. F. & RobinsoIL, W. S., eds), pp. 533-543, Academic Press, New York. Dunn, J. & Studier, F. W. (1973a). I’TOC. Nat. Acarl. Sci., U.S.A. 70, 1559-1563. Durln, J. & Studier, F. W. (1973b). J’TOC. Xat. Acud. Sci., U.S.A. 70, 3296-3300. Grohmann, K., Smith, L. H. & Sinsheimer, R. L. (1975). &ochemistry, 14, 1951-1955. Hayashi, M. N. & Hayashi, M. (1972). J. ITirol. 9, 207-215. ,Jorpcnsen, S. E., Buch, L. B. & Nierlich, D. P. (1969). S&ewe, 164, 1067-1070. Lebowitz, P., Weissman, 8. M. & Radding, C. M. (1971). J. Biol. Chem. 246, 5120-5139. Lpe, A. S. & Sinsheimer, R. L. (1!)74). I’roc. Sat. Acad. Sci., U.S.A. 71, 2882 2886. Lindqvist, B. H. & Sinsheimer, R. L. (1967). J. ;\lol. Bid. 30, 69.-80. Mait,ra, U., Nakata, Y. & Hurwitjz, .J. (1967). J. Viol. Ch,ern. 242, 4908%4918. Maizels, N. M. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3585-3589. Minklcy, E. G. & Pribnow, D. (1973). J. 3201. Biol. 77, 255-277. Roscnberp, M., Kramer, R. A. & Steitz, J. A. (1974). J. Mol. Bid. 89, 777-782. Seoburg, I’. H. & Schaller, H. (1975). J. 12Zol. Bid. 92, 261-277. Smith, L. H. 8: Sinsheimer, K. L. (1976a). ,J. Mol. Biol. 103, 681-697. Smit,ll, L. H. & Sinsheimer, R. L. (1976b). .J. Mol. Biol. 103, 699-710. Smith, L. H., Grohmann, K. & Sinsheimer, R. L. (1974). N&eic Bcids Res. 1, 1521-1529. Soavo, C., Nucca, It., Sala, E., Viotti, A. & Galante. E. (1973). Ew. J. Biochevz. 32, 392-400. Sugimoto, K., Okamoto, T., Sugisaki, H. & Takanalni, M. (1!)75). Nature (London), 253, 410-414. Sugiura, M., Okamoto, T. 8r. Ta.kanami, M. (1970). Xatlcre (London), 225, 598-600. Zain, B. S., Dhar, R., Weissman. S. M., Lebowitz, I’. 8: Lewis, A. M., Jr (1973). J. Viral. 11, 682-693.