Transcription termination sites in the major leftward operon of coliphage lambda

VIROLOGY

88,

252-262 (1978)

Transcription

Termination Sites in the Major Leftward Operon of Coliphage Lambda

JOHN S. SALSTROM*

WACLAW

AND

SZYBALSKIT

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 Accepted April 4, 1978 A transcriptional mapping technique was employed to determine the locations of leftward transcription termination sites within the pL-att region of bacteriophage h. Lysogens in which phage or host mutations blocked N-mediated antitermination were used. The results are consistent with the presence of three major termination sites, designated tL1,tL2,and tL3, which map at about 71.1 (between genes N and ral), 68.9 (likely within gene ~111) and 64.4 (within gene exe) XX units, respectively, measured from the left terminus of the mature X DNA. The tLI terminator is about 80% efficient, while the tI,Zand hn terminators are over 90% efficient. The locations of other possible terminators, including one near the n&L site and another within the int gene, are also discussed. INTRODUCTION

The expression of an operon can be negatively controlled by (i) interference with the initiation of transcription at the promoter-operator region, or by (ii) blocking the progress of transcription at promoterdistal termination sites. Both control mechanisms are operative in the Escherichia coli phage h. The present communication focuses on controls over termination of transcription. In very early in uiuo transcriptional studies with h it was observed that in the absence of gene N product (achieved by N mutations or by the addition of protein synthesis inhibitors), leftward and rightward RNA synthesis was limited (Taylor et al., 1967) to regions proximal to the PI. and pa promoters (Kourilsky et al., 1968; Kumar et al., 1969; Heineman and Spiegehnan, 1970; Szybalski et al., 1970). The in vitro work of Roberts (1969) indicated that h RNA synthesis was arrested at specific points when the host protein rho was present. For the major leftward A operon, Roberts (1969) found that the rho-terminated in vitro transcript was about 12 S, or about 250,000 daltons, a size which roughly * Present address: The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138. t To whom reprint requests should be sent.

matched that of the in uiuo synthesized 12 transcript (about 225,000 daltons; KouriIsky et al., 1968) deriving from the same region. The designation tL for the specific terminator of this transcript was proposed (Kumar and Szybalski, 1970; Szybalski et al., 1970) and its location judged to be at about 71.8 %h on the physical h map, based on the size of 12 S RNA and the 73.5 %A position assignment for the startpoint of leftward transcription, SL (Szybalski and Szybalski, 1974). The purpose of the present study was to determine the position of tL with greater precision by the transcriptional mapping technique of Hayes and Szybalski (1973). In addition, we attempted to establish the existence and positions of two other possible N-sensitive transcription termination sites observed in uiuo: one, a short distance downstream from tL, was indicated by higher molecular weight shoulders on the size distribution curves for hN- leftward RNA (observed in the experiments of both Kourilsky et al., 1968 and Lozeron et al., 1976); the other, thought to be between genes xis and exo, was indicated by the genetic studies of Inokuchi and Dove (see Appendix) with a X phage strain carrying a deletion of the gum-tL region (see also Szybalski, 1972). The experiments were performed under 252

0042~6822/78/0882-0252$02.00/O

Copyright 0 1978by Academic Press, Inc. All rights of reproduction in any form reserved.

TRANSCRIPTION

TERMINATORS

253

IN LAMBDA

conditions designed to block or reduce the the earlier assignment. The ~LZ terminator could correspond to the rho-independent possible effects of other control mechaterminator observed in vitro by Roberts nisms that might obscure or prevent simple (1975). The t~3 terminator likely corretermination events, including h DNA replication, N-mediated antitermination, and sponds to the polar site observed by Inoknegative control by the product of gene WO. uchi and Dove (see Appendix). The results of our study show that there MATERIALS AND METHODS are at least three major TVtermination sites Strains. All bacteriophage, bacterial that are operative in uiuo. Terminator ~~~ corresponds to the original TV of Kumar and strains and lysogens are listed in Table 1. was selected Szybalski (1970), indicated by the data of AninL4N7N53cI857cro27P3 Roberts (1969), but was found to map at a on QR48(434) at 39°C as a Fec+immh recombinant from a cross between Xbioposition a little farther downstream than

TABLE

1

BACTERIOPHAGE, BACTERIAL STRAINS AND LYSOGENS EMPLOYED Relevant Bacteriophage +8Osus2psu3 AbZc-

characteristics

imm80cI’; carries su3 suppressor b2 region deleted

hb2imm21

b2 deletion, imm21

hbiol6Ared3 Abio386cI857 hbio7-20 Abio72 Abioll Abiol

Aatt-inl Aatt-xis Aatt-xis Aatt-exe A&t-bet A&t-bet

Abio275cI857 Xbio250cI857 AbiolOcI857 Abio243cI857 Xbio232cI857 AbioN2-lnin5

Aatt-gum (57.3-68.8)” Aatt-gam (57.3-69.2)” Aatt-EalO (57.3-70.75)” Aott-ral (57.3-71.55)” Aatt-ral (57.3-72.05)” A&t-N (57.3-73.0)”

Abio3h-lnin5 AninIAN7N53imm434c-P3

Aatt-s,. (57.3-73.5)” Ah-h (67.9-72.2) K,

Atrxt,., AninL4N7N53cI857cro27P3

(57.3-58.9)” (57.3-60.3)” (57.3-62.7)” (57.3-66.1)” (57.3-68.1)” (57.3-68.2)”

(67.9-72.2) N-, P-

ninL4, N-, cro-, Pi

AbiolON7N53cI857cro27P3 Bacterial strains and lysogens w3350 W335O(AcI857P3).-, W~~~O(AN~N~~CI&~P~).=I W3350(AN7N53~1857cro27P3).11 W3350(Abio7-20nutL3c1857P3).-I W3350(AninL4N7N53c1857P3).=1

Aatt-EalO,

N-, cro-, P

0 ::o (xN+P-) sun (AN-P) sd (AN-CT-O-P) sue (AN+nutL-P-) s~~(Mtwt,.~N-F)

W3350(AninL4N7N53c1857cro27P3).PI

su’(AAtl2-k.lN-cro-P-)

W3350(AninL1N7N53c1857P3),-1(+80sus2su3+)

suO(AAt,.>.t,,lN-P-)

SKB178groN785(AcI857029P3).-, W335O(AN5imm21cIts5P3),-, QR48(434)

su”gro!V(AN+O-P-) su0(AN&imm21P-) su2+recA~( imm434)

= See Szybalski

and Szybalski

imm434, P

(1974).

(5~3~)

Source or reference Andoh and Ozeki (1968) H. Loseron; Kaiser and Jacob (1957); Kellenberger et al. (1961) G. Kayajanian; strain Ab265 of Kellenberger et al. (1961). H. Greer; Manly (1970) Adhya et al. (1974) Kayajanian (1968) H. Greer; Manly et al. (1969) H. Grew; Manly et al. (1969) H. Greer; Wollman (1963) and Manly, et al., (1969) H. Greer; Kleckner (1974) J. &hell; Signer et al. (1969) Signer et al. (1969) Signer et al. (1969) H. Greer: Signer et al. (1969) F. R. Blattner; Kayajanian (1968) and Blattner and Dahlbere (1972) F. R. Blattner; Kayajanian (1968) H. Inokuchi; M. Fiandt, W. Szybalski and W. F. Dove (unpublished data); see Appendix H. Inokuchi; M. Fiandt, W. Szybalski and W. F. Dove (unpublished data); see Appendix This study; see “Materials and Methods” J.S.S. H. Lozeron; Campbell (1961) Salstrom and Szybalski (1978) Salstrom and Szybalski (1978) Salstrom and SzybaIski (1978) Salstrom and SzybaIski (1978) This study; see “Materials and Methods.” This study, see “Materials and Methods.” This study, see “Materials and Methods.” M. Furth H. Inokuchi and this study J.S.S.; see Signer and Weil (1968)

254

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AND

lON7N53cI857cro27P3 and XninL4N7N53imm434c-P3. We constructed the W3350(hninL4N7N53cI857P3) ($180~~3)double lysogen because, for unknown reasons, we were unable to select viable ninL4N+ phages to examine the effect of N’ protein on termination of transcription at tL3; su3 suppresses both the Nam7 and Nam53 mutations to permit growth of heteroimmune ANN- phage types on this lysogen at all temperatures tested (from 30 to 42°C). All monolysogens were constructed as described by Salstrom and Szybalski (1978) (see Table 1). Methodology of RNA-DNA hybridization analysis. Isolation of separated phage DNA strands was by the poly(U,G)-CsCl technique of Hradecna and Szybalski (1967), as described in more detail by Bavre et al. (1971). Growth and preparation of heat-inducible lysogens for pulse-labeling with [3H]-uridine (New England Nuclear Co.) were essentially as described by Hayes and Szybalski (1973). Isolation of total hspecific leftward RNA by hybridization to Xb2 or to Xb2imm211 strands and analytical RNA-DNA hybridization were performed according to Bevre and Szybalski (1971) and Bevre et al. (1971). Other experimental details are described by Salstrom and Szybalski ( 1978). RESULTS

To locate the positions of leftward transcription termination sites in phage h, we employed the technique of transcriptional mapping (Hayes and Szybalski, 1973) in which the amount of purified phage-specific leftward RNA hybridizing to Z-strand DNA of each of several Xbio substitution phages was plotted versus the physical map position of the righthand deletion endpoint (the lefthand endpoint of each is fixed at the att site). In this way, we examined [3H]-pulselabeled leftward phage RNA synthesized in uiuo at various times after thermal induction of lysogens genotypically or phenotypically defective in the phage gene N function, a condition that permits transcription termination. The N+ condition permits leftward transcription to proceed through nearly the entire length of the pL-att region as early as l-2 min after induction, as indicated by the

SZYBALSKI

continuously rising N+ curve (see Fig. 1 including the insert; the possible reasons for small deviations from linearity are considered in the “Discussion”). However, under each of the three genetic conditions conferring the N- phenotype (the N- or nutL- phage mutations, or the groN host mutation), pr-initiated RNA synthesis is limited to the promoter-proximal region of the operon, as indicated by a rather abrupt decrease in the slope of the curves. This decrease can be interpreted as termination of RNA synthesis within the gum-N region. The location of the experimental points indicates the possible presence of more than one discrete termination point. In Fig. 1, the experimental points were connected with three straight-line segments, the middle segment being drawn through the bio250 and biol0 points. A simple interpretation is that the extrapolated break points correspond to two discrete terminators, designated tL1 and tm in Fig. 1. However, other interpretations compatible with the data shown in Fig. 1 could include that actually more than two discrete termination points exist within the bio250-bio10 interval, or that the termination points are not at all unique and that termination occurs with high probability anywhere within the interval. The results specified below, together with the data shown in Table 2 and Fig. 2, and the results obtained with mutants in which the tL1 or tL2 terminator regions were deleted (see “Discussion”) appear to strengthen the simple interpretation. The tL1 terminator. In Fig. 1, as drawn, the first “breakpoint” in each curve occurs between the biol0 and bio243 endpoints near the left terminus of the imm21 substitution. The data presented in Fig. 1 are based on three experiments, with 1-min pulse labeling starting at 1 min after thermal induction of the lysogens. However, analogous experiments were also done with [3H]RNA synthesized by heat-induced lysogens (see Table 2) pulse labeled at various other times (1 min pulses beginning at 2,3, 5, 9, 10, 14, 15 and 20 min after induction). There is remarkable consistency within these transcriptional mapping data, permitting us to assign an approximate position for the first breakpoint at 71.1 + 0.2% on

TRANSCRIPTION

TERMINATORS

NN

att hi

*is

! +I

a’.

I’

N c_ I ^

FIG. 1. Transcriptional mapping of leftward termination sites. Lysogens were pulse labeled with [3H]-uridine at 1-2 min after thermal induction at 42’C. RNA was extracted and leftward RNA purified by pre-hybridizing to the isolated 1 strands of hb2 DNA, followed by RNase treatment and by inactivation of RNase (Bgvre and Szybalski, 1971). RNA was eluted and each RNA sample was analyzed by rehybridizing to the 1 strands of hbio phages. The ordinate represents the percentage of total TCA-precipitable [3H] counts (1% = 2 to 7 X lo4 c.p.m. depending on [3H]RNA input) that hybridized to the hbio strains whose endpoints are shown below the abscissa in relationship to the physical (lowermost line) and genetic (heavy line) maps. Also shown are the relative positions of thepL promoter, the SL transcription startpoint and the TV, t~2, and tL3 transcription termination sites (see “Results”). The three lower curves were obtained by connecting the experimental points with straight line segments, with the vertical dashed arrows pointing to the two breaks representing the TV and t~2 termination sites (for tw see “Results” and Fig. 3). Most of the experimental points are averages of duplicate hybridization values for one [3H]RNA preparation; the duplicate values differed by less than 5%. The shaded arrows represent the corresponding RNA transcripts with the arrowheads indicating termination, and the reduction in width corresponding to reduced rate of transcription. The left terminus of the imm21 substitution is indicated near the right edge of the Figure. The insert in the upper lefthand corner is a contracted-scale drawing included to show the data

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the physical map (see Table 2). We interpret this breakpoint as the first major leftward transcription termination site, tag, which reduces pl-initiated transcription by a factor of about five (determined by calculating the ratio of the slopes on either side of tag). This interpretation is supported by the following data and discussion. The tL2 terminator. A second more distal breakpoint in each of the three lower curves in Fig. 1 (between the bioll and bio250 endpoints) indicates the possible presence of a second termination site. To ascertain the position of this second breakpoint with greater precision, the [3H]-labeled RNA from the NN- lysogens was pre-hybridized to the I strands of himm21b2 DNA to exclude the bulk of RNA deriving from the pL-N-tLI region (see below abscissa of Fig. 2). When RNA purified in this way is analyzed as in Fig. 1, only one breakpoint is observed (see Fig. 2). We interpret the latter as indicating the second termination site, tL2. An analogous single breakpoint at tL2 was observed during transcriptional analysis of AN-2+zm21PP [3H]RNA, which on the one hand indicates that tL1 termination is absent in this strain and on the other strengthens our conclusion about the existence of tL2 as an independent termination signal (Table 2 and J.S.S., unpublished data). From these and other data presented in Table 2 we assign to tL2 the approximate position of 68.9 f 0.2% on the physical map, likely within gene ~111 (see Discussion). In the experiments presented in Figs. 1 and 2, the tL2 termination appears to be greater than 90% efficient. The tL3 terminator. To ascertain the existence and location of the possible subsequent transcriptional termination signal(s) downstream from the tL2 region, we employed the special mutant, XninM, isolated by Inokuchi and Dove (see Appendix) and referred to earlier as A4 (see Fig. 3 of Szybalski, 1972). Deletion ninL4 appears to remove a segment carrying the tL1 and tL2 sites, as mapped above. Fig. 3 shows the results of transcriptional mapping for obtained for hIV in its entirety relative to ANN-. N’ (a-0): W335O(hcI857P3); NN(A-A): W3350(hN7N53cI857&‘3); n&L(O-----O): W335O(hbio72OnutL3cI857P3); groN(U): SKB178groN785(hcI857029P3).

SALSTROM

AND TABLE

Lysogen (relevant genotype)” 1. 2. 3. 4. 5.

SZYBALSKI 2

MAPPING OF THE h, kZ AND, tL3 TERMINATION SITES Number of ex- Prehybridized tlJd tL2 perimen& to I strands of:’

n&L-PgroN(O-P-) NN-P-“_ NN-cro-P-

4 2 6 5 7 7 8 6 6

6. -“7. NT, immZlP8. ninL4NN-P 9. ninL4NWcro-Pm

hb2 hb2 hb2 Xb2imm21 Xb2 Ab2imm21 hb2imm21 hb2 hb2

71.1 + 0.1 71.1 + 0.1 71.2 f 0.3 71.1 f 0.2

68.4 68.9 68.8 68.8 68.5 69.0 68.8

f + + + f + f

t1.3 OAd 0.2” 0.3” 0.2’ 0.5” 0.1’ 0.1’

64.4 + 0.2’ 64.4 * 0.F 64.4 + 0.3p

n See Table 1. * Number of independent RNA samples pulse labeled with [“HI-uridine for 1 min at various times between 1 and 21 min after thermal induction. No systematic difference between the early and late labeling times was observed. For details see “Materials and Methods,” and Figs. 1-3. ‘See “Materials and Methods.” ‘Three straight line segments were drawn through experimental points (1) between pi and biol0, (2) for bio250 and biol0, and (3) between att and bio250. The positions of the intersections between these segments were determined in relation to the physical map of h (see also Fig. l), and the averaged values, including standard deviations, are specified. e Positions of intersections between two straight line segments corresponding to (2) and (3) in footnote CE(see also Fig. 2). ‘The tLI termination is absent and the efficiency of tL2 is reduced to about 80% in hN;limm21 (Salstrom et al., in preparation). The positions of tL2 and tw were derived in a manner analogous to that described in footnote d. fl Determined as shown in Fig. 3.

.tt 60

65

70-&h

FIG. 2. Transcriptional mapping of the tLZtermination site. [3H]-pulse-labeled RNA was prepared as in Fig. 1, but was purified by pre-hybridizing to the 1 strands of hb2imm21 followed by RNase treatment to eliminate the bulk of the p&h RNA (represented by the open dashed arrow). For other details see Fig. 1. The broken lines represent W335O(hN7N53cI857P3) pulse labeled at 1-2 min (0) or 3-4 min (A) after induction at 42’C; the solid lines represent W3350(hN7N53cI857cro27P3) labeled at 1-2 min (0) or 20-21 min (0).

AninL4N- strains (cro+ and cro-) and for the control strain in which N function is restored by the su3 suppressor. Transcription initiated at pi under either N+ or N-

conditions appears to proceed unencumbered through the ninL4 deletion-fusion point; unlike the N+ curve, however, the Ncurve has a breakpoint between the bio7-20 and bio72 endpoints, near the left end of gene exo (as positioned by Szybalski and Szybalski, 1974 and unpublished). From the data presented in Fig. 3 and from data similarly obtained for other 1-min pulselabeling times after induction (for both cro+ and cro- derivatives of AninIANN-Pand for hNzl-imm21P; see Table 2), we estimate the position of this ~~~ breakpoint to be 64.4 f 0.3% on the physical map. Judging from the ratio of the slopes on either side of the ~~~point, we estimate that ~~~ termination is nearly absolute under Cro+ conditions, but is somewhat reduced (about 90% efficient) under Cro- conditions (see Fig. 3). DISCUSSION

The tL1 and tL2 terminators. The present data are more consistent with the presence of two terminators of in viva transcription in the N-gam region, rather than only one tL terminator as originally postulated (Kumar and Szybalski, 1970). This interpreta-

TRANSCRIPTION

TERMINATORS

5-6 MIN

att

int

xis

PI 60

iTcxo

bet

tL3

! 1

N ‘LPL

65 % ~&+$-yy, :

FIG. 3. Transcriptional mapping of the tL3 termination site. In the main figure, leftward RNA from W3350(XninLUV7N53~1857P3) (0, 0) or from W3350(XninL4N7N53cI857P3)(~80sus2su3+) (0) pulse labeled at the times indicated, was isolated by pre-hybridization to the 1 strands of hb2 DNA and was analyzed as described in Fig. 1. The ti.z-~111~ti.1segment of h DNA deleted in these strains is indicated below the physical and genetic maps and the distance between the bio72 and bio3h-I endpoints on the abscissa (as well as on the maps) is shortened accordingly. Transcription through the deletion-fusion point up to the ri.~ site under N conditions is indicated below the abscissa by the shaded arrow of uniform width. The insert shows analogous data for W3350(hninL4N7N53cI857cro27P3) at the times indicated. Here the deletion-fusion point is indicated by the symbol “A”. Both in the main figure and in the insert, the experimental point corresponding to the deletion-fusion represents the hybridization values for a hbio having an endpoint within the deleted interval. For other details, see Figs. 1 and 2.

tion is based on (i) the consistency of the calculated positions for the breakpoints corresponding to tL1 and tL2 (Table 2 and Fig. l), (ii) the considerable distance between these points (about 2.2%X units, i.e.,

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about 1000 base pairs), (iii) the presence of only one prominent breakpoint at the tL2 position in the experiments shown in Fig. 2 and Table 2, lines 4 and 6, and (iv) analogous results obtained when the tL1 region is deleted (Salstrom, Fiandt and Szybalski, in preparation) or substituted by imm21 DNA (Table 2, line 7). The position of tL1 at 71.l%h is about 0.7%h units farther downstream than the earlier less precise estimates based on the 12 S value assigned to the pL-N-h, RNA (see Roberts, 1969 and Szybalski and Szybalski, 1974), and is consistent with the leftmost estimate of Fiandt et al. (1974). This places tL1 very close to the position of the left imm21 boundary at 71.l%h (Szybalski and Szybalski, 1974). Termination in the tL1 region is about 80% efficient (see Fig. 1). The pr,-initiated transcription, which escapes through tL1 under N- conditions, appears to be nearly completely blocked at tL2, positioned at 6&9%h, just upstream of gene gam and likely within ~111(see below). The cro gene product appears to have little or no effect on the efficiency of tL1 and tL2 termination at the times tested (up to 21 min after induction), consistent with the earlier results of the Hu et al. (1975). This result and its relationship to the Fed phenotype (Court and Franklin, as cited by Franklin, 1971) are discussed elsewhere (Hu, SalStrom and Szybalski, in preparation). As mentioned in the Introduction, the in vitro initiated leftward transcript observed by Roberts (1975), which was substantially larger than 16 S RNA, could have resulted from termination at tL2 (assuming a molecular weight of about 750,000 daltons for this RNA, instead of 600,000 suggested by Roberts, 1975). The tL3 terminator. The third termination site, tL3, was observed when the tL1 and tL2 sites were removed by the ninL4 deletion. RNA synthesis initiated at pi under either N+ or N- conditions proceeded unencumbered through the ninLA deletion-fusion point, but was blocked with nearly absolute efficiency at the tL3 site under Nconditions (see Fig. 3). There is no obvious reason, however, why under Cro- conditions the observed efficiency of the tL3 termination site should be reduced. The tL3

258

SALSTROM

AND

site might correspond to the rho-independent termination site observed in vitro by K. Carlson (in preparation). Whereas the tL1 termination site appears to be located between two genes, the tL2 and tL3 sites may each lie within genes: tL2 within gene ~111(at 68.9%) because the righthand endpoint of deletion b1319 lies within gene ~111 and has been mapped at 68.9 +: 0.5% (Henderson and We& 1975); and tL3 within gene exo (at 64.4%A) because the righthand endpoint of deletion b522 lies within gene exe and has been mapped at 64.3%h. Thus, we suspect that transcription termination at tL2 and tL3 may occur through somewhat different mechanisms than at h, but nonetheless can be neutralized by the N antitermination function. Other possible terminators in the tL,-tL3 region. Oppenheim et al. (1977) have presented translational data interpreted as evidence for a termination site between genes bet and exo (at 66% on the physical map and which they refer to as h). The data presented in Fig. 3 for hninL4 are consistent with a constant rate of transcription from pi through this region to the tm site (at 64.4%) under N- conditions, regardless of the availability of Cro function, although the absence of more experimental points between the bio72 and bio7-20 endpoints makes it impossible to judge whether there is only one tL3 terminator or more transcription termination points in this region. However, the position of tL3 within gene exo would nonetheless cause depression of Exo protein synthesis relative to Bet protein under N- conditions, and therefore would be consistent with the results of Oppenheim et al. (1977). At face value, the data presented by Oppenheim et al. (1977) could be interpreted as evidence for four leftward termination sites. The reduced ability of hN- phages (relative to AN”) to synthesize the protein for each successive gene as assayed in their system, could be indicative of separate termination signals between each two genes, rather than being due (as they suggest) to some nonspecific deterioration (under Nconditions) in the ability of RNA polymerase to synthesize mRNA as it proceeds away from the pi promoter. Within this concept, the positions of each of the three

SZYBALSKI

leftward transcription termination sites deduced from our data could all be corroborated by their data. However, it is important to note that, while our system detects transcription terminators, their assay system also detects any post-transcriptional control leading to decreased protein synthesis. We would suggest here for the sake of clarity that our system of nomenclature ( tL1, tL2, tm, etc.) be used exclusively for terminators of transcription and that effects observed only at post-transcriptional or translational levels be designated in some other fashion. A possible tm terminator. As can be seen in Fig. 1 and as observed by Hu et al. (1975) and by Salstrom and Szybalski (1978)) transcription in thepi,& region in hN- mutants is reduced by a factor of two or more when compared with that observed for AN+ lysogens. The most obvious interpretations are (1) that there is an N-sensitive terminator (with only a two-fold effect) preceding the N gene, (2) that the N product increases the efficiency of initiation of transcription at pi (Kumar et al., 1970 and Szybalski, 1974), or (3) that degradation of the pL-tL1 RNA is more rapid under Nthan N+ conditions. The latter two explanations seem unlikely, at least in simple form, since we observed a depressed level also in the induced of pL-tL1 transcription hN+nutL- lysogens (Fig. 1; see also SalStrom and Szybalski 1978). If such a pL-proximal terminator exists, it would be quite analogous in strength to the first terminator for rightward transcription tR1 located near the right imm434 boundary, as deduced from the in vitro data of Roberts (1969) and the in viva transcriptional analysis of Kumar and Szybalski (1970). In analogy to the close proximity of tR1 to the postulated n&R site (see Rosenberg et al., 1978), there might be a similar relationship between n&L and the postulated tLo site. Transcription in the int-xis region. Under ~111~N+ conditions (in hnid.4, we observed that the leftmost point of the N+ curve is low (Fig. 3), indicating a small deficit of RNA in the int gene region. This result was also seen for all other 1-min pulse-labelling times tested (through 21 min after induction of XninL4). On the

TRANSCRIPTION

TERMINATORS

other hand, such a deficiency was observed only very early after induction of hcIII+N+ lysogens (Fig. 1) and disappeared within 5 min after induction (see Salstrom and Szybalski, 1978). The simplest interpretation is that the m-initiated N-antiterminated transcription is partially impeded within the int region and that this deficiency is masked under cIII+N’ conditions (within 5 min after induction) by the cII/cIII-dependent pi-initiated int gene transcription (see Katzir et al., 1976; Court et al., 1977; Pilacinski et al., 1977), except at the very early times (e.g., l-2 min; see N” curve in the Fig. 1 insert) when levels of ~111(and/or ~11) protein(s) may be insufficient to permit full PI-initiated transcription of gene int. Processing and degradation of the piN-tL1 RNA. Examination of the shape of the N+ curve in Fig. 1 reveals a deficit of thepL-proximal RNA in the biolO-sL region, which was shown by Lozeron et al. (1976, 1977) to be a result of a selective degradation initiated by the endonucleolytic action of RNaseIII. Lozeron et al. (1977) have shown that this partial loss of the pL-proximal RNA is eliminated in the RNaseIIIdeficient hosts. To rule out effects of RNaseIII on the patterns of leftward transcription presented here, these experiments should be performed in an RNaseIII- host. However, we believe that the results of such experiments would not significantly alter out present interpretations. ACKNOWLEDGMENTS We are most appreciative of the gift of the ninL4 deletion phage from Dr. W. F. Dove. We thank Ms. J. KriIl for the expert technical assistance, Mr. M. Fiandt for the unpublished meaurements of the ninL4 deletion, and Dr. Elizabeth Szyhalski for the critical reading of the manuscript and unpublished physical mapping of gene eno. This work was supported in part by a pre-doctoral fellowship to J.S.S. from the National Institute of General Medical Sciences and by NC1 Grant No. CA-07175. REFERENCES ADHYA, S., GOTTESMAN, M. and DE CROMBRUGGHE, B. (1974). Release of polarity in E. coli by gene N of phage h: Termination and anti-termination of transcription. Proc. Nat. Acad. Sci. USA 71.2534-2538. ANDOH, T. and OZEKI, H. (1968). Suppressor gene su3+ of E. coli, a structural gene for tyrosine tRNA. Proc. Nat. Acad. Sci. USA 59, 792-799. BLAITNER, F. R., DAHLBERG, J., BOETTIGER, J.,

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