JOURNAL OF BACTERIOLOGY, Feb. 1991,

p.

1554-1560

Vol. 173, No. 4

0021-9193/91/041554-07$02.00/0 Copyright 0 1991, American Society for Microbiology

Characterization of the Late-Gene Regulatory Region of Phage 21 HWAI-CHEN GUO,t MARK KAINZ, AND JEFFREY W. ROBERTS*

Section of Biochemistry, Molecular and Cell Biology, Biotechnology Building, Cornell University, Ithaca, New York 14853 Received 2 April 1990/Accepted 17 November 1990

A segment of Escherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages K and 82. The leader RNA is about 45 nudeotides long and consists essentiafly entirely of sequences encoding the p-independent terminator that is the putative target of the antitermination activity of Q21. Like the corresponding regions of A and 82, the 21 late-gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase. The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in arns, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of K and 82. There is little evident similarity in the primary sequences of the three Q genes.

Growth of the lambdoid phages is regulated at the level of transcription termination (for a review, see references 4 and 17). The transcription antiterminators encoded by bacteriophage A genes N and Q (5) activate expression of the phage early and late genes, respectively, by allowing Escherichia coli RNA polymerase to transcribe through terminators located upstream of regulated genes. Phage X Q protein (QX) activates phage late-gene expression by allowing elongation of the constitutively expressed 6S leader RNA, which starts at the phage late-gene promoter (PR') and stops in the absence of QX at a strong p-independent terminator (tR') that precedes the late genes (9). The nucleic acid sequences required for QX function (qutX, for QX utilization) span the RNA start site and include the promoter elements and part of the adjacent transcribed region (25, 28). The late-gene regulatory region of phage 82 has a similar structure and mechanism of action (6); it also encodes a late leader RNA, named 82a. Other lambdoid phages, such as 21, 480, and the Salmonella typhimurium phage P22, also encode small RNA transcripts like X 6S that arise from corresponding regions of their genomes and that are putative leader sequences for late-gene expression (21). Q proteins are genome specific in function, despite their presumably common mechanism of action. QX acts on the phage X late-gene promoter or the nearly identical phage P22 late-gene promoter (8, 18) but not on the phage 82 late-gene promoter. Similarly, phage 82 Q protein (Q82) acts on the phage 82 genome but not on the phage X genome (6, 27), although it may share specificity with phage +80 Q protein (22). Therefore, Q proteins presumably interact with at least two transcription components: with DNA or RNA, to account for the genome specificity, and with RNA polymerase, to account for their action as antiterminators at various sites distant from the recognition and interaction site. The details of the molecular interactions between the Q proteins and these specific sequences are unknown. However, part of the mechanism of Q protein function is known and furthermore is apparent simply in the interaction of RNA polymerase with the late-gene promoter: transcription *

Corresponding author.

t Present address: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138. 1554

pauses for several minutes in vitro about 16 nucleotides (nt) after initiation at the X late-gene promoter PR' (9). During this pause, QA modifies RNA polymerase into an antiterminating form (9, 28). Similarly, phage 82 has pause sites at +15 and +25 in the beginning of its late operon (27), at which Q82 acts. Deletion and point mutations that abolish the pauses also abolish the ability of Q proteins to mediate antitermination (7, 28). To study the generality and specificity of Q proteinmediated antitermination, we have characterized the lategene regulatory region of another lambdoid phage, 21. We report the DNA sequence of a segment containing the phage 21 Q gene, its late-gene promoter, the 21a leader RNA, and the p-independent terminator of this leader RNA. We show that phage 21 has a genetic organization and an antitermination control mechanism similar to those of phages A and 82, despite having no obvious homology to their Q genes or protein sequences or to DNA encoding their leader RNAs. Phage 21 Q protein (Q21) is active as an antiterminator in vitro and is specific for the 21 late-gene region.

MATERIALS AND METHODS Materials. All materials were of the highest purity available and were purchased from commercial sources, unless otherwise stated. Restriction enzymes were purchased from New England BioLabs, Bethesda Research Laboratories, IBI, and Amersham; T4 polynucleotide kinase, T4 DNA ligase, and exonuclease III were purchased from New England BioLabs; the Klenow fragment of DNA polymerase I was purchased from Bethesda Research Laboratories; S1 nuclease, calf intestine alkaline phosphatase, and T4 DNA ligase were purchased from Boehringer Mannheim; reverse transcriptase was purchased from Molecular Genetics Resources; RNase T1 and lysozyme were purchased from Worthington; radiolabeled nucleoside triphosphates were purchased from Amersham; Taq DNA polymerase was purchased from USB; nucleoside triphosphates and dideoxynucleotides were purchased from Pharmacia; synthetic oligonucleotide linkers were purchased from New England BioLabs; rifampin and isopropyl-o-D-thiogalactoside (IPTG) were purchased from Sigma; SeaPlaque agarose was purchased from FMC BioProducts; and low-melting point agarose was purchased from Bethesda Research Laboratories.

VOL. 173, 1991

PHAGE 21 LATE-GENE REGULATION

1555

(a) 21a

21

I

I

RV

I

I

I

I

I

I I

la

I

ii

I

RV

I

I

X CH S

R

I Cos

I I

A

A A VCVVB

CV

C

X

A

6SyXR

I

X R

I

S NVR

NIX,

I

C

cos

C

x

1 kbp

(b) 3.9

21

4

I. 21405.9

FIG. 1. Restriction and heteroduplex maps of the rightmost genomic segments of phages 21 and X. (a) Some restriction sites in the two rightmost EcoRI fragments of phage 21 DNA are shown. The corresponding restriction site map of phage X DNA is shown for comparison. Sites for restriction enzymes are indicated as follows: A, AvaI; B, BamHI; C, ClaI; G, BglI; H, HindIll; N, NarI; R, EcoRI; S, Sall; V, EcoRV; and X, XmnI. The filled boxes in the DNA segments of the restriction maps indicate the homologous regions between phages X and 21 determined on the basis of the heteroduplex shown in panel b. The coding regions of genes 0, P, Q, S, and R as well as late-gene leader sequences 21a and 6S are also indicated. (b) Heteroduplex map for phages X and 21 corresponding to the regions shown in panel a; the map is taken from Simon et al. (23; used by permission). The numbers represent the percent length of A.

Oligonucleotides were synthesized at the oligonucleotide synthesis facility of the Cornell Biotechnology Program. RNA polymerase, purified by the method of Burgess and Jendrisak (la) as modified by Lowe et al. (11), and NusA protein, purified as described previously (7), were provided by J. Goliger (our laboratory). Bacterial and phage strains. The construction of phage K cI857(QSR)21hX was described previously (21). Plasmids were constructed and maintained in E. coli HB101 [F' lacjP hsdS20 (rB mB) recAJ3 ara-14 proA2 lacYl galK2 rpsL20 (Smr) xyl-5 mtl-l supE44 X-]. M13mplO, M13mpll, and their derivatives were propagated in E. coli JM101 [A(lac-proAB) supE thilF' traD36 proAB+ lacIZAM15]. E. coli JWR208 (F' lacIWL) (9) transformed by pKD301 was used to purify Q21. Plasmids. The plasmids used and their sources were as follows: pHG11O (this study), pHG210 (this study), pKD301 (this study), pXY306 (28), pA82(51)t. (7), pYH101 (a lowcopy-number plasmid compatible with pBR322, expressing resistance to spectinomycin, and containing the gene Q82) (7), pXY401 (like pYH101, except that the EcoRI fragment containing Q82 was substituted by an EcoRI fragment from plasmid pUV5.Q2 containing QA (28), pKD303 (like pYH101, except that the EcoRI fragment containing Q82 was replaced by the polymerase chain reaction-amplified fragment containing Q21) (see below). DNA subcloning of the phage 21 QSR region. Standard cloning manipulations were performed as described by Maniatis et al. (12). Ligation reactions usually were performed in SeaPlaque low-melting-point agarose as described by Struhl (24). Restriction digestions were done as specified by manufacturers. For restriction mapping and sequencing of the 21 QSR region and transcription mapping of the 21 late promoter, two EcoRI restriction fragments of A cI857(QSR)21hX (21), 21B (-3.7 kb) and 21C (-3.3kb), were isolated, end filled with the Klenow fragment, connected to EcoRI linkers (because 21C has the cos end), and cloned into EcoRI-

digested, 5'-dephosphorylated pBR322 (yielding pHG11O and pHG120, respectively). For construction of pHG210, pXY316 (28) was cut with HindlIl, end filled, and digested with EcoRI to yield a 4.4-kb Hindlll (blunted)-EcoRI vector. pHG11O was cut with Sall, end filled, and digested with EcoRI, and the 400-bp Sall (blunted)-EcoRI fragment was cloned into the HindlIl (blunted)-EcoRI vector described above, yielding pHG210. For subcloning of Q21 into expression plasmids, a 564-bp DNA fragment containing the Q21 gene and terminating in EcoRI restriction sites was amplified from pHG320 (a derivative of pHG11O containing the 1,680-bp EcoRI-EcoRV fragment of 21 DNA) by the polymerase chain reaction. Oligonucleotide primers for amplification (5' CGGAAT TCCTCAGTAAAAACCATTCC 3' and 5' CGGAATTCCT CAATGATGTCAACACG 3') ended in EcoRI restriction sites and matched 21 DNA sequence segments between nt 212 and 230 and nt 742 and 760, respectively, (see Fig. 2). The amplification reaction consisted of 1.2 nM pHG320 DNA, 200 nM each oligonucleotide primer, 175 ,uM deoxynucleoside triphosphates, and 10 U of Taq DNA polymerase in 10 mM Tris hydrochloride-2.5 mM MgCl2-50 mM KCI0.01% gelatin (pH 8.3). DNA was amplified through 15 cycles of 60 s at 93°C, 40 s at 45°C, and 100 s at 72°C and 1 cycle of 60 s at 93°C, 40 s at 45°C, and 10 min at 72°C. The 564-bp amplified fragment was isolated following electrophoresis through low-melting-point agarose, cleaved with EcoRI, and reisolated from low-melting-point agarose. Plasmid pKD301 was constructed by inserting the amplified DNA fragment into the unique EcoRI restriction site adjacent to the modified phage T7 Al promoter controlled by the lac repressor in pUHE21-2 (1). pKD301 was used to overexpress Q21 for purification. Plasmid pKD303 was constructed by replacing the EcoRI fragment containing the Q82 gene in pYH101 (7) with the amplified fragment bearing the Q21 gene. pKD303 is compatible with pHG210 and was used as the source of Q21 for in vivo Q21 specificity assays. DNA sequencing. Fragments were end labeled with reverse

1556

GUO ET AL.

J. BACTERIOL. 60 120

240 300

~ TA AGGCT A AGPACG A M G I R E L N L T K E Q H

E W L N 360 G MWG GS 1 P 1JGU3It1A AAIA G A W V Y S G R L E K R M S 420 AATG AC A M~AGOGO AGCTAAGI A GGOG TAG S V I A K F M E S V E P G R V M T R P M AI~T ~GMCIP1V G W L E L W

480

C N D

D D G

M L I S

Q V V

D S V

M Y I D 540

ACAAAGC K K A A S Y

C rCX AC A PTA cmACaTr OG=IcClA F G I

Y H R

GCATAA ACATG I Q K P S L

Ourr L F M

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XcATcG G1CGCAAGAC CTCTAAG V A R P

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I K H

+1 CA

K H A I

u;i

600

A Gc-3ra L C R G G G R 660

P G TXC G TG M GLXGAST CICAASAI A T C R R E V D E I L N A S 720

GATTTXA G1I"ITTAAACCPGA MCGrGLW I Y P

V L D S

A F K

-35

AAATCAAA

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G

AIOQ

N R K

R V E K

-10

780

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V A .

+18

830

I CYGY: CDaTTSTSTSATT 890 I Ep A GSAGTC T C TG=C GlAA= 950 MClC XAMA tCrcA2T' GWGIC tAS1UnWL 995 CG~AAGTGIO 7rFACGTk AGCZCAAA CMM

FIG. 2. Sequences of the phage 21 Q gene and 21a RNA coding region. The nucleotide sequence of the RNA-like strand of the phage 21 Q gene and the predicted amino acid sequence of Q21 are shown. DNA encoding the 45-nt 21a RNA is in boldface type. The late-gene promoter -35 and -10 consensus sequences (underlined), the start site of transcription (+1), and the transcription pause site (+18) are indicated. Arrows indicate the dyad symmetry sequences of t2l that are typical of p-independent terminators. A 16-bp sequence which contains a Shine-Dalgarno consensus sequence (indicated by asterisks) and is identical to the corresponding region in A is indicated by wavy underlining. The point at which extensive similarity to A ends is indicated by @. Sequences identical to A beyond the recombination point (at A bp 43,068) are underlined; these sequences are in a homologous region of the heteroduplex between phages 21 and A (Fig. 1).

transcriptase and sequenced as described by Maxam and Gilbert (13) or cloned into M13 and sequenced as described by Sanger et al. (19). Both strands were sequenced for the interval from bp 150 to bp 995 that included the entire phage 21 region (see Fig. 2). RNae T1 mapff of the 21a leader sequence and paused

transcpt. t-3 P]ATP (350

RCi)

was vacuum dried and

dissolved in transcription buffer. A Salb-EcoRI restriction fragment from pHG11O contng the phage 21 late promoter and terminator was used as a DNA template in a 10014 transcription reaction without rifampin. Transcription was stopped after 20 min by phenol extraction, and 5'-endlabeled RNA was isolated by polyacrylamide gel electrophoresis. The paused RNA (18 nt) and the terminated 21a RNA (-45 nt) were excised from the gel, extracted in TE buffer,

and subjected to RNAse T1 partial digestion or limited alkaline hydrolysis as described by Donis-Keiler et al. (2). In vitro transcription reactions. For preparation of the DNA template used for in vitro transcription reactions, DNA plasmids purified with CsCl gradients were digested with the appropriate restriction enzymes, sticky ends were filled in, and large vector DNA was removed by polyethylene glycol precipitation as described by Lis (10). The template was a SalI-EcoRI restriction fragment containing the phage 21 late-gene promoter and terminator of the leader RNA (t2l) or a HindIII-EcoRI fragnent containing the phage X late-gene promoter and terminator (9). Transcription stopped at t2l resulted in the -45-nt 21a RNA; transcription through t2l resulted in a 230-nt readthrough RNA. Templates for transcription mapping of the 21a coding region were purified by extraction from low-melting-point agarose. Transcription reaction mixtures (25-,ul final volume) containing 50 mM Tris chloride (pH 7.9), 0.1 mM EDTA, 1 mM dithiothreitol, 80 mM KCl, 200 ,uM ATP, 200 p,M GTP, 200 p,M CTP, 25 p,M UTP, 2.5 p,Ci of [Ia-32P]UTP, approximately 1 to 2 nM DNA template, 10 to 20 nM purified RNA polymerase holoenzyme, 150 nM NusA protein, and 400 nM Q protein, when applicable, were preincubated for 10 min at 37°C. Synthesis was initiated by the simultaneous addition of MgCl2 and rifampin to final concentrations of 5 mM and 10 p,gfml, respectively, and allowed to continue for S min (see Fig. 6) or 10 min (see Fig. 3 and 4). Synthesis was stopped by the addition of 75 p.l of transcription stop buffer (0.66 M Tris chloride [pH 7.9], 15 mM EDTA, 0.25 mg of tRNA per ml), and the reaction mixtures were placed on ice. For transcription pausing experiments, a larger restriction cocktail was preincubated, transcription was initiated, and 25-,l aliquots were removed and added to 75 pi of stop buffer at various times. RNA was extracted with 100 ILI of phenol-chloroformisoamyl alcohol (1:1:0.04), ethanol precipitated, and resuspended in 3 ,ul of formamide loading dye containing 0.01% bromophenol blue-0.01% xylene cyanol. RNA was resolved by electrophoresis through 0.4-mm-thick 50% urea-polyacrylamide sequencing gels and visualized by autoradiography. Radioactivity in resolved RNAs was measured on dried gels with a Betascope, and percent readthrough was calculated as molecules of readthrough RNA divided by the sum of terminated and readthrough RNAs. in vivo alacokinase assays. E. coli HB101 (F' lad") cells (obtained from K. McKenney) carrying different promoterterminator and Q protein source plasmids were grown in L broth containing 0.1 mg of ampicillin per ml. For induced samples, IPTG was added to 1 mM to cells at an optical density at 650 nm (OD650) of 0.2. Growth was continued for 1.5 h before sampling; the final OD6" of the cultures was 0.6 to 0.8. Toluene-treated cells were assayed in duplicate as described by McKenney et al. (14). Units of galactokinase are given as nanomoles of galactose phosphorylated per minute per millimeter of cells at an ODwo of 1.0. Plasmid copy number was examined by restriction digestion of plasmid DNA isolated from an aliquot of cells equal to that assayed for galactokinase activity no more than a twofold difference among transformants was observed. Purification of Q21. Q21 was evident as a major protein in extracts of cells containing pKD301 and grown in the presence of ITG. Q21 was purified the same way as Q82 (6), except that the Bio-Gel HPI column was omitted and chromatography on phosphocellulose was followed by chromatography on MonoS. The final purity was estimated to be 70%. Nucleotide sequence accession number. The GenBank ac-

VOL. 173, 1991

PHAGE 21 LATE-GENE REGULATION

21a RNA

+18 pause RNA

H 0 0.5-1 2 0 0.5 1 2 0 1

DNA

(unitngRNA)

21

... 11.5 2z.

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1557

.......

tiine(nn

-2a

00.

..

RTV

a5: 6*58 3

A6S

(230)

7

AOL4

_* RT

(390)

_

...;_ _......

t.-

-.

Zia,

U 4_ A G_

(45) :~~

36

MW

lwwllwmw'.WWW

A1

+16

proucs prdue wit difrn amut ofRaeT:sson

pause

Two dupicate sts are hown fo 21a RN. Lane represnts.th

asdecIbed in MNateaalyss and Mthods ThesequencofAan thepNAue transcript is8shon auonaigrmo the TfRN right.n cleavagesathgune nuclotidesicare indicated byow arows RNAa Undgete RNAe is alreensoth designated.

cession number for new sequences reported in this paper is M58702.

RESULTS Restriction and transcription mapping of the 21a RNA coding region. A 7-kb DNA fragment of phage X cl857(QSR)21hX, in which the X late-gene regulatory region is replaced by that of 21, encodes the putative phage 21 late-gene leader RNA 21a (21). To characterize this region, we cloned into pBR322 the two EcoRI fragments that comprise the 7-kb segment from gene 0 to the right end of the phage and mapped them by restriction analysis (Fig. la). The map agrees well with the structure of heteroduplexes between X and 21 in this region (23) (Fig. lb). The restriction sites are conserved in homologous regions (Fig. la, filled segments) and different elsewhere (Fig. la, open segments). By transcribing restriction fragments in vitro, we mapped the 21a coding region to the 380-bp SalI-EcoRI fragment (see below); this site corresponds in location to the X 6S RNA coding region but is in a nonhomologous segment (Fig. la). DNA sequencing of the promoter segment and RNase T1 mapping of 21a RNA. The 380-bp Sall-EcoRI fragment was cloned and sequenced. Consistent with the RNA mapping described below, we found a consensus -35 hexamer and an acceptable -10 hexamer, followed downstream by sequences appropriate for a p-independent terminator (Fig. 2). The predicted transcript size, about 45 bases, is consistent with the mobility of the in vitro transcription product determined by denaturing polyacrylamide gel electrophoresis. The 21a late-gene leader sequence was confirmed by RNase T1 partial digestion, which cuts RNA at the 3' side of guanidine residues; limited alkaline hydrolysis provided a

FIG. 4. Pausing during the synthesis of the phage 21 late-gene leader RNA. An autoradiogram of a transcription time course with the native 21 or X DNA template is shown. Transcription in the absence of Q protein was stopped at the times indicated. The pause at + 18 of phage 21 (with a minor band at + 19), the pause at +16 of phage X (actually a +16-+17 doublet), 21a RNA, X 6S RNA, and readthrough (RT) RNAs are indicated, with nucleotide lengths in

parentheses.

size ladder (Fig. 3). Partial site-specific degradation of the transcripts occurred at pyrimidines during isolation in the absence of RNase T1, particularly following a pair of UMPs (Fig. 3). When this background is taken into account, the pattern of cleavage products was that expected from the DNA sequence (Fig. 3, arrows). End labeling of the 21a

RNA with [_y-32P]ATP indicated that transcription initiation with ATP. The transcriptional start site was determined on the basis of the site of pause and the size of the paused RNA (Fig. 3 and 4) and was uncertain by 1 or 2 bases. The putative p-independent 21a terminator has the canonical form of a simple terminator, i.e., a run of U residues preceded by a G+C-rich dyad symmetry element that could form a stem-loop structure with a AG' of about -21 kcal (ca. -88 kJ)/mol (Fig. 2). It is remarkable that the terminator is essentially the entire transcript. This terminator is very efficient in vitro (Fig. 4) and, apparently, in vivo occurs

(data not shown).

RNA polymerase pauses at +18 of the putative late-gene leader RNA. Grayhack et al. (9) showed that QX recognizes RNA polymerase paused at +16 of the late-gene transcript and modifies it into an antiterminating form. Likewise, transcription complexes paused at +15 and +25 are substrates for Q82 (7, 27). We found that RNA polymerase also pauses in transcription initiated from the 21 late-gene promoter, at about + 18. Figure 4 shows transcription products made at intervals of up to 8 min during synchronized single-round transcription from the 21 late-gene promoter.

1558

J. BACTERIOL.

GUO ET AL.

0c

0

a)

0)

Amp

on

FIG. 5. Structure of pHG210. Bam, BamHI; HIII, Hindlll; Rl,

EcoRI; Smal, SmaI. The 18-mer disappears with a half-life of 2.5 min, and there is a corresponding increase in 21a RNA. Mapping of the paused transcript by RNase T1 partial digestion revealed a pattern of cleavage products identical to that obtained from the leader RNA (Fig. 3), showing that the paused RNA polymerase is indeed initiated at the promoter that we identified. The pause site of +18 was inferred from the mobility of the RNA in relation to that of the transcript paused at +16 (Fig. 4); the size of the latter was determined previously (9). The paused transcript migrates slightly more slowly than the + 18 position of the nuclease-digested RNAs, consistent with its lack of a charged 3' phosphate group. Cloning and sequencing of the 21 Q gene. Both the A and the 82 Q genes are encoded immediately upstream of their leader sequences. Since this seemed likely for phage 21 as well, we sequenced about 1 kb of DNA upstream of the 21a RNA coding region. An open reading frame (ORF) 486 bp long was identified (Fig. 2). A consensus Shine-Dalgarno sequence exists immediately upstream of the start codon AUG (Fig. 2, asterisks). A 16-nt segment near the start codon has the same sequence as the corresponding region of A (Fig. 2, wavy underlining). We demonstrated (see below) that the 486-bp ORF indeed encodes Q21. Interestingly, this ORF is located immediately downstream of the site at which near-identity to A DNA ends (Fig. 2, @) (nt 233 in Fig. 2 corresponds to A bp 43,068) and immediately upstream of the -35 element of the 21a RNA promoter. On the basis of the sequence, Q21 is a basic protein (net charge, +9) with a molecular weight of 18,000. Activity and specificity of Q21. To demonstrate that the ORF encodes a function with the regulatory activity expected of an antiterminator, we extracted the ORF by the polymerase chain reaction and cloned it into the expression

vector that we had used previously for QA and Q82, yielding pKD303 (see Materials and Methods). For the test, a second plasmid containing a qut site with its associated promoter and one or more terminators placed upstream of the galactokinase gene is used. Q protein function is detected as galactokinase activity resulting from transcription initiating at the qut-associated promoter and proceeding through the terminator(s). In this test, QX is specific for qutA and Q82 is specific for qut82. Table 1 shows that this specificity extends to Q21: the activity of each of the three Q proteins is specific for its cognate qut site. Q21 is an antiterminator in vitro. The definitive proof that a protein is an antiterminator is its activity in a purified transcription system in vitro. We found that an expression plasmid carrying the putative Q21 gene downstream of the T7 Al promoter made a polypeptide, detectable in stained sodium dodecyl sulfate gels of cell extracts, that migrated with mobility appropriate for a protein of 18 kDa, the mass of Q21. We purified this protein to about 70%o homogeneity by methods similar to those used for QX and Q82, and we assayed its activity in antiterminating transcription (Fig. 6). Reaction mixtures contained 150 nM NusA protein, which is required for efficient activity of QA, although we did not test whether NusA is required for efficient activity of Q21. The positions of the terminated transcripts (21a and A 6S) and readthrough RNAs are shown in Fig. 6. Purified Q21 increases readthrough on the 21 template from 2.6 to 23% and has a slight effect on the template, increasing readthrough from 2 to 7%. On the other hand, QX increases readthrough on the X template from 2 to 30% and has no effect on the 21 template. We conclude that Q21 is an antiterminator that acts specifically in vitro, although its has detectable activity on

DNA

as well as on

21 DNA.

DISCUSSION We have shown that phage 21 has the same genome organization and encodes the same antitermination mechanism as do phages A and 82; it is likely, but has not been shown, that Q21 regulates late-gene expression for phage 21. In all three phages, gene Q is separated from the late genes by a promoter and terminator that encode a leader RNA or putative leader RNA. The homology of phage 21 with the other phages, as well as the activity of Q21 that we have found, indicate that phage 21 gene Q encodes an antiterminator that allows transcription initiated from the 21a RNA promoter to read through a strong p-independent terminator, t21, and proceed into the late-gene operon. Like qutA and qut82, qut2l encodes an early transcription pause at nt 18 of the late-gene operon. No obvious secondary structure in the nascent RNA at the pause site is apparent, nor does a region

TABLE 1. Activities and specificities of three Q proteins in vivoa Activity (U of galactokinase) of Q protein provided by expression plasmid: qut site on assay plasmid

A 82 21 None

QX QX

No Q gene No Q gene

Expt 1

Expt 2

Expt 1

Expt 2

42 4 10 4

27