J. Mol. Riol. (1992) 228,488~50.5

Characterization

of Bacteriophage T7 RNA Polymerase by Linker Insertion Mutagenesis

Lyndon Gross, Wei-Jia Chen and William T. McAllister Morse Institute

for Molecular Genetics, Department of Microbiology and lrnmurcoloyy of New York, Health Science Center at Brooklyn University 450 Clarkson Avenue, Brooklyn, NY 11203-2098, V.S.A.

State

(Received 20 March

1992; accepted 25 June

1992)

Thirty-four mutants of phage T7 RNA polymerase (RNAP) were generated by linkerinsertion mutagenesis and characterized with respect to their ability to carry out various of mutants with interesting biochemical steps in the transcription cycle. A number properties were identified. These include: (1) Mutant RNAPs that are catalytically active but that bind weakly t,o a T7 promoter: one of these mutants is affected in a region of the RNAP that exhibits homology with the sigma subunit of Escherichia coli RNAP. Another is affected in a region that has been previously implicated in the discrimination of T7 versus T3 promoters (Joho. et al., 1990). (2) Mutant RNAPs that can bind to the promoter but are transcriptionally inactive; some of these RNAPs lack catalytic activity. others are catalytically active but are unable t.o initiate productive transcription at a T7 promoter. Among the latter class of mutants are enzymes that appear to be weakened in their ability to melt open (or to remain associated with) double-stranded DNA; these RNAPs make only abortive initiation products and are unable to proceed to the formation of a productive elongation complex. The mutations causing this phenotype affect regions of the RNAP that exhibit homology with the catalytic site of DNA polymerase I (Delarue et aE., 1990). (3) A C-terminal insertion mutant with properties similar to a previously characterized “foot” mutant (Mookhtiar et al., 1991). This RNAP appears to be defective in the very early steps of transcription and may be unable to translocate and/or empty the active site. (4) A mutant that is transcriptionally active, but is unable to complement the growth of T7 gene 1~ phage. This phenotype may result from disruption of a function of the RNAP that is distinct from its role in RNA synthesis.

Keywords: DNA polymerase

I; active site; sigma subunit; abortive initiation

1. Introduction

DNA

melting;

independently of its role in viral replication and allowing the isolation of large quantities of protein for physical and biochemical analysis (Davanloo et al., 1984; Tabor & Richardson, 1985). Furthermore, the enzyme has been crystallized, suggesting that detailed structural information may soon be available (Sousa et al., 1989; Chung et al., 1990). Here, we present a preliminary characterization of T7 RNAP by means of linker-insertion mutagenesis, in which two codons were inserted at various positions in the polymerase gene through the use of a kanamycin resistance (KmR) cassette that was developed specifically for this purpose (Chen et al., 1992). In all, 34 RNAP mutants were generated in this manner. The mutants were characterized in vivo with respect to their ability to synthesize functional messenger RNA, their temperature sensitivity, and their ability to complement T7 gene lp phage.

The RNA polymerase that is encoded by bacteriophage T7 is attractive as a model for studying the structure and function of DNA-dependent RNA polymerases (RNAPT). Compared to other RNAPs, the phage enzyme has a relatively simple organization, and consists of a single species of protein of 98,000 M, that is able to carry out accurate transcription in the absence of any auxiliary proteins (Chamberlin & Ryan, 1983). The gene that encodes the phage RNAP (gene I) has been cloned and expressed in bacteria, permitting genetic manipulation and characterization of the RNAP t Abbreviations used: RNAP, RNA polymerase; IAA, indole acrylic acid; DTT, dithiothreitol; Km, kanamycin; Cm, chloramphenicol; w.t., wild-type; nt, nucleotide(s); DMSO, dimethyl sulfoxide ITC, initial transcribing comlplex; EC, elongation complex.

488 0022-2836/92/220488-l

8 $08.00/O

0

1992 Academic

Press Limited

Linker

Insertion

Mutagenesis of T7 RNA Polymerase

The mutant RNAPs were also characterized in vitro in a series of reactions designed to assess their ability to carry out various steps in the transcription cycle. These assays included: ability to bind specifically to promoter-containing DNA fragments, or non-specifically to random DNA fragments; ability to carry out abortive and productive initiation from a T7 promoter; ability to carry out nonspecific (promoter-independent) catalytic activity; and ability to recognize a T7 termination signal. To facilitate screening, initial assays were performed using cell-free extracts. Mutant RNAPs of interest were subsequently isolated by a simplified protocol involving one-step chromatography on a column of phosphocellulose. A number of RNAP mutants with interesting biochemical properties were identified. Some of these enzymes appear to be blocked at key steps in the transcription process. Although further studies will be required to understand the nature of the defects that are responsible for these phenotypes, the results suggest a number of regions of T7 RNAP as candidates for future analysis by site-directed mutagenesis.

2. Materials and Methods (a) Bacteria

and plasmids

pWJCl2, a trp promoter (GenBlock, To construct Pharmacia) was inserted into the HindHI to BarnHI interval of pBR322; transcription from the trp promoter is directed in the same orientation as the tet gene. The T7 map gene present in PAR1219 (Davanloo et al., 1984) was inserted into the BamHI site of pWJC12 under control of the trp promoter, to give pWJC16. Subcloning of T7 gene I into pWJC12 was necessary because PAR1219 contains an ApaI site (important in the mutagenesis protocol) within the Zac UV5 promoter. (b) Indirect determination of RNAP activity in vivo by measuring the level of chloramphenicol resistance A loopful

(10 ~1) of a fresh overnight culture of coZi BL21(DCAT4) (Dunn et al., 1988) carrying the desired plasmid was streaked on the surface of an LB agar plate containing 50 pg ampicillin/ml (LB + Amp; Sambrook et aZ., 1989) and a filter paper strip (Whatman, 3MM, 60 mm wide), which had been immersed in a solution of 5 mg chloramphenicol/ml and drained, was laid across the inocula. After 16 h at 37”C, the zone of inhibition of growth of the culture on each side of the strip was measured. Temperature sensitivity was determined by spotting 5 ~1 of culture onto LB+Amp plates containing 0, 2, 10 or 50 pg chloramphenical/ml and incubating at 25°C 30°C 37°C and 42°C. Escherichia

recognized by growth of the inoculum on both sides of the phage lane. (d) Preparation

by

A loopful (10 ~1) of an overnight culture of BL21(DCAT4) carrying the desired plasmid was drawn across the agar surface of a petri plate through a lane of x104 T7 gene l- phage particles (T7 4107; Studier & Moffatt, 1986). Complementation is recognized by growth of the bacterial culture on the proximal side of the phage lane and lysis on the distal side; failure to complement is

of cell extracts

LB media (5 ml) containing 50 ,ag ampicillin/ml were inoculated with l/100 volume of a fresh overnight culture and incubated at 37°C until the culture had reached an absorbance at 600 nm of @6 (-3 h). Indole acrylic acid (IAA, 62 mM) was added, and after 4 h bacteria were harvested by centrifugation (3000 g, 10 min). The pellets 5 ml harvesting buffer were washed once in (50 mM-Tris.HCl (pH 80), 2 mrvr-EDTA, 20 mM-NaCl, 1 miw-dithiothreitol (DTT)) and then resuspended in 05 ml of buffer containing 200 mM-NaCl and 100 pg eggwhite lysozyme/ml (Boehringer-Mannheim). Cells were lysed by 4 cycles of freezing and thawing (-70°C room temperature) and the viscosity was reduced by sonication (2 x 30 s, Kontes microprobe, full power). Bacterial debris was removed by centrifugation (15,000 g, 10 min) and the clarified samples were stored in 100 ~1 portions at - 20°C. (e) Partial

puriication

of mutant RNA Ps

Bacterial cultures (50 ml) were propagated and induced as described above. The cells were washed, resuspended in 1.5 ml of harvesting buffer containing 61 mMphenylmethylsulfonyl fluoride, and lysed as described above. Ammonium sulfate (92 M) and polyethyleneimine (95% (w/v)) were added, and after 15 min at 4°C the sample was clarified by centrifugation at 15,000 g, 5 min. 1 : 10 in buffer A The supernatant was diluted (20 mnn-potassium phosphate (pH 7.7) 1 mM-EDTA, 5% glycerol, 1 mM-DTT), clarified by centrifugation, and applied to a 2 ml column of phosphocellulose (Whatman Pll) in a Biorad Poly-Prep column. The column was washed with 8 ml of buffer A containing 200 miw-NaCl, and the RNAP was eluted with buffer A containing 400 mm-NaCl, applied in 95 ml volumes. Samples (10 ~1) of each fraction were spotted onto a sheet, of Whatman 3 MM paper, which was then stained with Coomassie blue to visualize the protein (Jorgensen et al., 1991). Peak fractions (usually the 2nd and 3rd fractions) were pooled. Glycerol (50%) and DTT (01 mM) were added, and the When analyzed by samples were stored at -20°C. SDS-PAGE, a single major band at the position expected for T7 RNAP was observed; the concentration of the RNAP in the partially purified samples ranged from 100 to 200 pg/ml, as judged by comparison of the stained bands with standard preparations. The enzyme preparations are substantially free from nuclease contamination (Gross, 1992). Highly purified preparations of wild-type RNAP (98,000 M, form) were prepared as previously described (Grodberg & Dunn, 1988). T7 RNAP specifically cleaved to the 80,000/20,000 M, form was prepared by incubation with extracts of E. coli HMS174, as described by Muller et al. (1988).

(f) Protein (c) Com,plementation of T7 gene l- phuge particles mutant RNA polymeruses

489

analysis

Samples (100 ~1) of cell cultures were analyzed by electrophoresis in 10% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE), as described by Studier (1973). (g) Preparation

of synthetic

DNA

DNA was synthesized on a Biogen DNA synthesizer and purified by chromatography on a Sep-Pak column

I,. hhss

490

et al.

Oligomer concentration was estimated (Waters). assuming a molar extinction coefficient (per base) of Oligomers were end labeled with E 260 -- 84 x 103 M-~. [y-32P]ATP using T4 polynucleotide kinase (Sambrook rt oligomers (250 np. non-templatca al.. 1989). Labeled strand) were mixed with an equimolar amount of unlabeled template strand in 30 ~1 of 200 mM-NaCl and heated to 90°C for 10 min. After slow rooling to room temperature, the sample was precipitated at - 70°C with 1.25 M-ammonium acetate, 7004 ethanol. washed in 70”;, and resuspended in 50 /11 ethanol. dried in vacua @Ol M-Tris-HCI. 1 mM-EDTA (TN). Double stranded (ds) oligomers were resolved by electrophoresis in 15yb (w/v) polyacrylamide gels in TBE (Sambrook et a,l.. 1989). localized by autoradiography, and eluted from the gel by the crush-soak method (Maxam $ Gilbert. 1979). The sample was again precipitated with ethanol and resuspended in 50 ~1 TE. The incorporation in a I ~1 sample was determined by counting in Liquiscint (Nat,ional Diagnostjics); -60 x lo3 dts/min (2 ng) of ds oligomers were used for earh gel retardation assaT. For use as templates in thanscription rea&ions. the ohgomers were not end labeled or gel purified. However, the extent of annealing was monitored by gel elect,rophoresis followed by staining with ethidium bromide.

The ability of RNAP to bind determined as described by Muller

DXA fragments was et al. (1988) using the

same promoter-containing oligomer and random oligomer as suggested by these authors. Binding reactions (20 ~1) 10 mw-potassium phosphate contained: (pH 7.8). 1 mM-EDTA, 4’?/6 glycerol. 8 mM-MgC1,. 40x 103 to 60 x lo3 cts/min ( -2 ng) of the appropriate oligomer, 1 pg RNase A (Sigma). 1 ~1 cell extract. and. where

indicated 1 pg calf thymus DNA. Following incubation

at

37°C for 10 min the samples were loaded directly onto a pre-elecbrophoresed 8% (w/v) polyacrylamide gel and run at room temperature at 1.50 V in Tris-borate/EDTA (TBE) buffer (Muller et al.. 1988). The gels were dried and visualized by autoradiography.

Figure 1. Mutagenesis of T7 RSAI’ gene. Linker insrrtion mutagenesis of the Ti RNAP gene (gene I) using the K,mR cassette in pWJ(:3 was carried out as described by (!hen et al. (1992) and discussed in t’he text,. Abbreviations are as follows: I’ frp. tr;u promoter; AtnpR. bka gene: KmR, kanamycin resist,ance; ori: (‘01 El origin of DNA replication

3. Results (i) Transcription

(a) Mutagene8ix

reactions

Unless otherwise indicated: reactions were carried out 37 “C for 15 min in a volume of IO @I containing: 20 mM-Tris. HCI 8 rnM-MgCl,, 2 rnM(pH 7.9). spermidine-HCl, 1 mM-DTT. 0.5 mM each of ATP, GTP. CTP and UTP (Pharmacia, Ultrapure). and 1 ~1 cell extract. Labeled substrates (a-32-P[rNTP]. New England at

Nuclear)

were present at a specific activity

of @2 Ci]

mmol. The synthesis of short run-off and abortive initiation products (Figs 4 to 6) was determined by modification of the procedures of Martin & Coleman (1987) and Martin et al. (1988) using 20 ng of promoter oligomer as template. Reactions to determine non-specific catalytic activity contained 100 ,ug rifampicin/ml. 5 pg synthetic polynucleotide template (Pharmacia) and Q.5 mM[z-~‘P]GTP. Runoff and termination assays (Figs 7 and 8) were carried out in 20 ~1 reactions containing 10 ng purified RNAP (see above) and 1 pg linearized plasmid DNA. Reactions were terminated by the addition of urea (3 M). EDTA (10 mM); bromophenol blue (O.Ol”/;, (w/v)) and xylene cyan01 (0.01 o!0 (w/v)). Samples were heated to 90°C for 2 min and analyzed by electrophoresis in 6 to 20% (w/v) polyacrylamide gels in 1 x TBE, 6 M-urea at 1800 V.

and

RNA polymerase

initial

screening mutants

of

pWJC16 (which carries T7 gene I) was subjected to partial digestion with restriction enzymes that generate blunt ends (Table I), ligated with the KmR cassette found in pWJC3: and transformed into BL21(DCAT4) (Fig. I. step (a)). This host. bacterium contains a chromosomal copy of the chloramphenicol acetyltransferase gene under control of a T7 promoter (Grodberg & Dunn, 1988; Dunn et al., 1988), and thus cells that contain a plasmid which provides functional T7 RNAP are chloramphenicol resistant (CmR), whereas cells with plasmids that do not provide T7 RNAP function are chloramphenicol sensitive (CmS). In the initial screen we sought, KmR. CmS clones in which the RNAP was inactive as a consequence of the KmR insertion. Plasmids of interest were cleaved with ApaI (to excise the KmR cassette), religated, and transformed again into BLBl(DCAT4) (step (b)). These operations result in the insertion of six basepairs (5’-GGGCCC-3’) that encode two amino acid

Linker

Insertion

Mutagenesis

of T7 RNA

Polymerase

491

Table 1 Predicted

changes in RNAP

structure Predicted amino acid ~eyuenre and secondary

Plasmidb

Insertion

site'

Wild-type

structured

Mutant

pLGl1

XmnI

(3194)

NTINIAKNDFSDIELA hshssccccchhhhhh

NTINIAKNGAEFSDIELA hshhhhcccccchhhhhhh

pLG19

AM

(3266)

ADHYGERLAREQLALE ccchchhhhhhhhhhh

ADHYGERLGAPREQLALEH cccttttccchhhhhhhhh

(3270)

DHYGERLAREQLALEH cchchhhhhhhhhhhh

HYGERLARGPEQLALEHE ccthhctccchhhhhhhh

pbV,JC17

HdII

(3281)

LAREQLALEHESYEMG hhhhhhhhhhhhhhhh

ERLAREQLGAPLEHESYEM hhhhhhhcccchhhhhhhh

pLG2H

BstITI

(34.56)

FEEVKAKRGKRPTAFQ hhhhhhhttccctshh

WFEEXKAKROPGKRPTAFQ hhhhhhhttcttccctshh

Km1

(3515)

KPEAVAYITIKTTLAC chhshhhhhsssssss

EIKPEAVAlfAliITIKTTLA ccchhhhhhhhhhtttsss

pWJC18

HUf?IlI

(3599)

AVASAIGRAIEDEARF ssshhhchhhhhhhhh

AVASAIGRGAPIEDEARFG ssssscctcccchhhhhhh

l'LG24

AM

(3644)

FGRIRDLEAKHFKKNV hhhhhhhhhhhhhhhh

GRIRDLEGaPKHFKKNVE hhhcccchhhhhhhhhhhh

&%I

(3810)

DSIHVGVRCIEMLIES tttthhhhhshhhhhh

EDSIHVGVGPRCIEMLIE ttttsttccctsssshhh

HincIl

(3836)

VACIEMLIESTGMVSL hhhshhhhhhhhhhss

CIEMLIESQPTGMVSLHR sssssstccccchssssc

pWJC30

RsaI

(4118)

RYEDVYMPEVYKAINI hhhhhhhhhhhhhhhh

EDVYMPEVWAEKAINIAQN htchhhhhhhhhhhhhhhh

pW,J('41

B&U1

(4295)

DMNPEALTAWKRAAAA hhhhhhhhhhhhhhhh

DMNPEALTGAPWKRAAAAV hhhhhhhcchhhhhhhhhh

pW,J('lS

HaelI

(4406)

KFANHKAIWFPYNMDW hhhhccctsstttsss

ANKFANHKG-IWFPYNMD hhhhhcttccctssstttt

pLG14

NlaIV

(4414)

FANHKAIWFPYNMDWR hhhccctssttttssc

FANHKAIWRALPYNMDWRG hhhhchhhhssccttttst

pWtJC33

RsaI

(4721)

LAFCFEYAGVQHHGLS hhhhhhhhhhhttttt

CFLAFCFEWAEAGVQHHGL thhhhhhhhhhhhhctttt

pLG20

AM

(4749)

GVQHHGLSYNCSLPLA hhhttttttttsssss

AGVQHHGLRGPYNCSLPIA hhhhcstttcttttsssss

pWJC23

HincII

(4845)

RDEVGGRAVNLLPSET hhhhttcsssccccss

DRVGGRAVdPNLLPSETV hhtttccccccccccsss

pLG12

XmnI

(4863)

AVNLLPSETVQDIYGI sssccccsssssssss

VNLLPSETGPVQDIYGIV sssscttcccsssssssh

pWJC24

HincII

(4902)

YGIVAKKVNEILQADA ssshhhhhhhhhhhhs

YGIVAKKVGPNEILQADA sssssstcccchhhhhhs

NlaIV

(4935)

LQADAINGTDNEWTV hhhhsccccccsssss

LQADAINGGPTDNEXVTV hhhssctcccccssssss

pLG18

Ah1

(4999)

SEKVKLGTKALAGQWL hhhhhhhhhhhhhhss

TGEISEKVKGRLGTKALAG chhhhhhtcccccsssshh

pLG17

NlaIV

(5088)

TLAYGSKEFGFRQQVL sssccccttthhhccc

SVMTLAYGGPSKEFGFRQ sssssstcccccttthhh

pWJC25

H&c11

(5110)

SKEFGFRQQVLEDTIQ ccttthhhccctttss

GSKEFGFRRAQQVLEDTI cccttthhhhhccsstts

pLG23

AluI

(5141)

QVLEDTIQPAIDSGKG ccctttssssctcssc

VLEDTIQPGAPIDSGKGLM cctttcccccccttttcss

pLG22

Ah1

(5206)

PNQAAGYMAKLIWESV ccchhhhhhhhhhhhh

QAAGYMAKGPLIWESVSV CCCSSstCCCCCCSsSSS

L. Gross et al.

492

Table 1 (continued) Predicted

Insertion

Plasmidb

‘41UI

pLG21

amino secondary

acid sequence structured

Wild-type

site’

(5240)

and

Mutant

WESVSVTWAAVEAMN

ESVSVTVVGAPAV-L

hhhhcchhhhhhhhhh

hhsssssscchhhhhhhhh

pLG27

BatUI

(5404)

KPIQTRLNNMFLGQFR cctssssssssstscs

EYKKPIQTOPRLNLMFLG tttcttsscctcssssst

pWJC34

RsaI

(5517)

APNFVHSQDGSHLRKT

SGIAPNFVBPHSQDGSHL ttcccctcccccttttcc

cctsscctttccctth

RsaI

pWJC35

NlaIV

pLGl5

XmnI

pLG13

B&l!1

pLG25

Raal

pWJC37

BstUI

pLG26

(5573)

(5613)

(5636)

(5655)

(5691)

(5816)

VWAHEKYGIESFALIH

TWWAHEKWAUGIESFALI

hhhhhttthhhhhhhc

hhhhhhhhhhhhhhhhhhh

FALIHDSFGTIPADAA

ALIHDSFGGPTIPADAAN

hhhhcttttccch

hhscttttcccsshhhhh

TIPADAANLFKAVRET

TIPADAANRALFKAVRET

ccchhhhhhhhhhhhh

cccchhhhhhhhhhhhhh

LFKAVRETMVDTYESC

ANLFKAVRQPETMVDTYE

hhhhhhhhhhthhhhh

hhhhhhcccccsssshhh

MVDTYESCDVLADFYD

DTYESCDVGPLADFYDQF

hhthhhhhhhhhhhhh

tttttttccccshshhhh

NLRDILESDFAFA

RDILESDFWPFA

ccssshhhhhhhh

ssssstttcchhh

“Designation of the mutant as in& signifies that the 6 base-pair linker is inserted within, or immediately after. the Sth vodon in 1’7 gene 1. %asmid designation for each mutant. ‘Enzyme used to generate the site of insertion; the position of the insertion in the gene I sequenre (Mofatt rt al. 1984) is indicated in parentheses. dThe predicted amino acid sequence in the region of the alteration is given; changes in the mutant enzyme are iudicatrd in boldface. Secondary structures were predicted by the program PROTLYZE (Scientific and Educational Software) using the algorithm of Gamier rt al. (1978): h, helix; t, turn; b, P-sheet; c, coil.

residues in all reading frames, and comprise an ApaI site. Since the sequence of the RNAP gene is known, the nature of the mutation may be deduced from the position of the insert (Table 1). To facilitate discussion, mutants are referred to as insN, signifying that insertion of the linker has occurred within, or immediately after, the Nth codon in T7 gene 1. (b) Integrity

and solubility

of the RNA

null

WT

ins783

ins/59

ins578

polymerase

To ensure that the reading frame of the target gene had not been disrupted, or that other gross disruptions had not ocurred during mutagenesis, the integrity of the mutant RNAP was determined by SDS-PAGE. An overexpressed protein having the expected mobility was observed for all mutants tested. However, the yield of RNAP varied among the isolates. In a number of cases the solubility of the mutant RNAP appeared to be compromised, and low amounts of enzyme were recovered in the soluble fraction (Fig. 2 and Table 2). Two of the mutant RNAPs (ins578 and ins589) are produced in low abundance; these RNAPs appear to be labile (as well as insoluble), and give rise to cleavage products that are readily detectable by Western blot analysis (not shown). Apparently, the modifications intro-

Figure 2. Integrity and solubility of mutant RNAPs. Cultures carrying the plasmids indicated were induced for 3 h with IAA. The cells were harvested by centrifugation. resuspended in buffer, lysed by 3 cycles of freezing and thawing in the presence of lysozyme, and the lysate was clarified by centrifugation. Equal volumes of the lysnte (T), the clarified supernatant (S) or the resuspended pellet (P) were analyzed by SDS-PAGE. Shown are null and wild-type (WT) samples, and examples of an insoluble mutant (ins783), a mutant of moderate abundance and partial solubility (in.y259), and a mutant of low abundance (ins578). * marks the position of T7 RNAP.

Linker Insertion

Mutagenesis of T7 RNA Polymerase

duced into these RNAPs destabilize the proteins and make them susceptible to protease degradation. In general, in the analysis that follows, we have not considered the biochemical properties of mutants that are insoluble or labile. The phenotypes of these mutants are provided in Table 2 in the hope that this information may prove useful in future investigations. (c) Activities

of the mutant polymerases in vivo

Cultures of BL21 (DCAT4) carrying plasmids of interest were streaked on the surface of a petri plate and a filter paper strip impregnated with chloramphenicol was laid across the various inocula. The zone of inhibition by the antibiotic was then measured (Table 2). Strikingly, 21 out of 34 mutants retained some RNAP activity in wivo, as evidenced by significant levels of chloramphenicol resistance. This result suggests that there are numerous positions in the RNAP that may be locally disrupted without abolishing transcription activity. None of the mutants exhibited a frank temperature-sensitive phenotype, as assessed by growth on media containing various concentrations of chloramphenicol at temperatures ranging from 25°C to 42°C (not shown). The CmR assay is apparently quite sensitive, and even mutants that produce low amounts of recoverable RNAP provide substantial resistance to chloramphenicol (see, for example ins589 and ins783, Fig. 2 and Table 2). (d) Complementation of T7 gene l- phage In addition to its role in late gene expression, T7 RNA polymerase is also involved in entry of the phage DNA into the cell, initiation of DNA replication, interaction with phage lysozyme, generation of mature DNA molecules from replicative concatamers, and packing of mature phage DNA into phage particles (Moffat & Studier, 1988; Roman0 et al., 1981; Moffat & Studier, 1987; Hinkle, 1980; J. J. Dunn personal communication). As these functions may involve specific protein-protein interactions, one might expect that some mutations would affect the capacity of the polymerase to carry out these activities without disrupting its basic transcription functions. To search for such mutations, the ability of mutant RNAP to complement gene 1- phage was determined by cross-streaking bacterial cultures through an inoculum of T7 4107, a deletion mutant that lacks all of gene 1 (Studier & Moffat, 1986). In all cases except one, mutants that retained transcription activity (as assessed by chloramphenicol resistance) were able to complement T7 4107. The single exception was ins691. Although this mutant gave rise to moderate levels of chloramphenicol resistance and normal production of RNAP (as judged by SDS-PAGE), it was unable to support growth of T7 4107. The failure of ins691 enzyme to complement T7 gene l- phage is not due simply to

493

its poor transcription activity, as other mutants that provide even lower levels of chloramphenicol resistance are able to complement the deletion phage (e.g. ins317; see Table 2). In related work with mutant subunits of E. eoli RNA polymerase, other investigators have observed trans-dominant lethal phenotypes in which RNAPs containing the defective subunit were defective in promoter clearance and thus interfered with the function of the wild-types enzyme (Kashlef et al., 1990). In the hopes of identifying ‘I’7 RNAP mutants with a similar phenotype, we determined the effects of overproduction of mutant RNAP on the replication of wild-type (w.t.) T7 phage particles. None of the mutants prevented phage replication, as determined either by cross-streak experiments or by a decrease in the efficiency of plating (data not shown). (e) Promoter binding To determine the ability of the polymerase to bind DNA, cell extracts were incubated with a 24 base-pair 32P-labeled oligonucleotide that contains a T7 promoter (specific binding) or a labeled oligomer that has the same base composition but does not contain a promoter (non-specific binding) (Muller et al., 1988). The protein-DNA mixtures were then resolved by electrophoresis in a polyacrylamide gel under conditions in which binding of the oligomer results in retardation of its mobility (Fig. 3). In the absence of competitor DNA, extracts from cells that contain wild-type T7 RNA polymerase are able to retard both types of oligomer. Addition of calf thymus DNA as a competitor prevents binding of the non-specific oligomer, but does not prevent binding of the promoter-containing oligomer. Titration of extracts that contain w.t. RNA polymerase by dilution in a null extract indicates that this assay can detect specific DNA binding over a loo-fold range of polymerase concentrations. All of the extracts tested have polymerase concentrations that lie within this range. Although they are inactive in viva, ins559, ins640, ins648 and ins881 proteins retain an ability to bind to the promoter, suggesting that these enzymes are blocked at some stage after promoter recognition and binding (Fig. 3, panel C; and Table 2). (ins815 and ins829 RNAPs also retained promoter binding activity, but have low solubility and were not considered further (see Table 2). Most of the enzymes that were active in vivo retained promoter-binding activity. However, a few of the active enzymes (e.g. those of ins33 and in.sl59) exhibited little or no promoter-binding activity in this assay (see Fig. 3 and Table 2). (f) Non-specijk

catalytic activity

The non-specific catalytic activity of T7 RNAP (that is, catalytic activity in the absence of a defined promoter sequence) can be measured as the synthesis of poly GMP (poly (G)) on a variety of

L. Gross et al.

494

A.

Non-specific Oligomer

Promoter Oligomer

II--++---++-+-+--+-+-

Bound DNA

T7 RNAP Competitor DNA

,(

B’ z

-2

o 8 ’

- x z RNAP Dilution

I2345

Free DNAI 2 3 4 5 6 7 8 9 IO

I 2

3

4 5 6

7 8 9 IO II 12

Figure 3. Promoter binding by mutant Rh’APs. Panel A, a 32P-labeled 24 base-pair oligomer that contains a T7 promoter (see Fig. 6, template I), or an oligomer that lacks a T7 promoter, were incubated with extracts from cells that express T7 RNAP (WT) or that lack T7 RNAP (null). Calf thymus DNA was present as a competitor where indicated. The samples were resolved by electrophoresis in an 8% (w/v) polyacrylamide gel and analyzed by autoradiography. The reactions shown in lanes 5 and 10 received no cell extract. Panel B, samples of an extract that contains T7 RPiAP were diluted in a null extract as indicated. and analyzed for ability to bind to the T7 oligomer in the presence of competitor D?U’A. Lane 1, null extract; lanes 2 to 5, w.t. extract, diluted as indicated. Panel C. samples of extracts containing the RNAP indicated were incubated with the T7 oligomer in the presence of calf thymus DNA and analyzed as described above. Results obtained with key enzymes are shown in this panel: other results are summarized in Table 2.

synthetic templates (Chamberlin & Ring, 1973). We assessed the ability of the mutant polymerases to carry out this reaction on poly(dC) (a singlestranded DNA template), poly(dI) * poly(dC) (a double-stranded DNA template of moderate stability), and poly(dG) . poly(dC) (a doublestranded DNA template of high thermal stability). Initial assays were carried out in the presence of poly(dC) and enzymes that were active on this template were then tested for activity on poly(dI). and (Table 2). poW(=) PMdG) PWdC) Enzymes that were inactive at the usual concentration of GTP (@5 nM) were also tested for activity at a tenfold higher concentration of GTP (5 mM) to determine whether they might exhibit, an abnormally high dependence on substrate concentration (not shown). On both poly(dC) and poly(dI).poly(dC) the w.t. enzyme makes abundant products of relatively large size (greater than 150 nucleotides (nt)). However, on the poly(dG). poly(dC) template the w.t. enzyme makes products of a much lower size distribution, and individual products terminating at each nucleotide position within the G-ladder may be resolved on appropriate gels (not shown). These results suggest that T7 RNAP has difficulty proceeding through the highly stable poly(dG) 1poly(dC) template. Similar observations have been made by Patra et aE. (1992). Most of the enzymes that we tested retained

catalytic activity. The only soluble RNAP that lacked activity (ins559) was also inactive in ,uiuo. as assessed by chloramphenicol resistance. Some mutants exhibited high levels of cat’alytic activity on poly(dC) but weak or little activity of poly(dG) .poly(dC). These enzymes may be unable to bind to double-stranded DNA, particularly if they also fail to bind promoter fragments (e.g. insZ59, ins841) However, some mutants that are inactive on poly(dG) * poly(dC) retain an ability bo bind to the T7 promoter (ins640, ins648, ins881). These enzymes may have a weakened ability to melt upon the DNA helix during initiation and/or elongation (see below).

(g) Initiation,

and the effects of dimethyl

sulfoxide

After promoter binding and the formation of an open complex, transcription is initiated by the synthesis of a few phosphodiester bonds. A stable elongation complex is not formed by T7 RNAP until a product of 8 to 12 nt is synthesized, and prior to stabilization the enzyme undergoes repeated cycles of abortive initiation in which short transcripts are synthesized and released (Martin r:f al., 1988; Ling et al., 1989). The ability of the mutant polymerases to carry out these abortive cycles of initiation and to synthesize a short (5 nt) runoff product using a 24 base-pair synthetic

++ ++ ++

+ + +

+ + +

+ + +

+ + +

+ + +

++ ++ ++

++ I ++ ++

+ + +

+ + +

+ + +

+ + +

+ + +

++ ++ ++

++ I ++ ++

+ + +

+ + +

+ + +

+ + +

++ 14-f ++

+ + +

+ + +

+ + +

+ + +

I

++ ++ ++

+ + +

+ + +

+ + +

+ + i

I

++ ++ ++

+ + +

+ + +

+ + +

I

++ ++ ++

+ +

+ + +

+ + +

I

+ + + + ++ +

+ I ++ -7 7 + +

++ + .+ + I-

+ + +

++ I if ++ ++ I ++++ ++

+ +

+ ++ ++ + -I‘

+++ 77;

+ ++ ++ + ‘I

I

+++ I+++1 +++

+ + +

+++ I+++; +++

+ + + I + f

++ I ++++ ++

+ I +

+ +

+

I

+ +

I

++

T+

I +

I +;

+ +

I

+ +

+++ ++

I

I

++ ++ ++ ++ ++ ++

+

T+T +++

+

I ++++ ++++

+ l+I++ +

+ +

if

++ +++

I +; 7,

+ + +

+++ +++

I++++,; ++

++++

+ + ++ + -_ ++ +

I

+++ +++ +++

I

+

I

I

I ++;+ +

++ ++ ++

I

+ + +

++ ++

+ + +

I + +

+ +

+++ I +++I +++

++ ++ ++

+ + +

++++ ++++I ++++

++ ++ ++

+ + +

+

I

I

I

+ + +

I

+i

+

+ ++

+++ ++++

++ ++

+ +

+

496

L. G-08s et al

Figure 4. Initiation of RR’A synthesis on a short sythetic promoter. Purified RNAP (98K) or extracts that lack (null) or contain T7 REAP (WT). were incubated with a synthetic 24 base-pair promoter that initiat.es with the sequence + 1 GGACI’ (see Fig. 6. t,emplate 1). Substrate rNTPs were present as indicated; the labeled rNTP is indicated in boldface. Products were analyzed b) electrophoresis in a 20% (w/v) polpacrylamide gel in the presrncar of 6 M-urea. followed hy a,utoradiography. IJane 1 shows a ladder of poly((:) products made by purified w.t. RNAP at. the ~$10 promoter. which initiat.es with the seyuencae + I GGG: the number of G residues in the product is indicated to the left of t,he lane. The identit,y of the products. as deduced from their size, the promoter sequence. and the pattern of label, is indicated to the right. (:onfirmation of certain assignments was aided by the use of incomplete mixtures of substrate and differential labeling (see inset. lanes 17 t,o 24). Products resulting from initiation of the REAP at the f 1 and + 2 position of the promoter are indicated.

promoter fragment was determined as described by Martin et al. (1988) (Fig. 4). In the presence of extracts that lack T7 RNAP, a single product is observed when GTP is the labeled substrate, but not when other rNTPs are labeled (Fig. 4, lanes 2 to 6). The identity of this product is not known. When w.t. RNAP is present, either in crude extracts (lanes 7 to I 1) or in purified form (lanes 12 to 16), the 5nt product GGACU is detected, as are abortive products 3 and 4 nt in length (GGA and GGAC). Low levels of products that arise by initiation at the +2 position (GA, GAC and GACU) are also observed. The abundance of these “false start” products appears to be independent of the structure of the DNA template around the start site, as an equivalent spectrum of products is seen from a promoter that is single-stranded in the initiation region (our unpublished results). In the presence of GTP as the sole substrate, purified RNAP makes abundant quantities of the initiating dinucleotide GG, and lesser amounts of poly (G) products of 3 and 4 nt in length (Fig. 4, lane 12). The ability of the phage RNAP to produce a ladder of poly (G) products up to 14 nt in length had been observed previously at promoters that initiate with the sequence + I GGG, and was attri-

Figure 5. Effects of DM80 on init.iation of t.ranxcLrip tion. Extracts t,hat wntzain the REAP int1icat.d WPI’C incubat,ed with a spnt~hetic 24 base-pair promoter as &scribed in Fig. 4. ,4114 rNTPs were present anti (iTI’ its was t,hr labeled substrate. I)MS0 was prrsrnt intlic~atrd

buted to slippage of the RNA product along thr template strand without translocat.ion of t,hr enzyme (“stutt.ering”; Martin pt rrl.. 198X: Cunningham et al., 1991). Our observation that) promoters that’ initiate with the sequence + I GG also permit st,uttering is in contrast to previous reports (Martin et al.. 1988). As only one nucleot~idr of the RNA product csould remain in associat,ion a#t t,he labter with thr template af%er slippage promoter, the stabilit,y of t’he complex is exp&ed to be great,ly diminished. This might, account for the lower level and shorter length of slippage products made at this type of promoter. Addition of ATP to the reaction suppresses production of the dinucleotide (Fig. 4. lane 13) as well as synthesis of the more extended Q-ladder at promotors that initiate with + 1 GGG (see Fig. 7; and Martin e6 al., 1988). We int’erpret these results to indicate that the formation of the first phosphodiester bond (and those that) arise by “stuttering”) is slower than the addition of subsequent nucleotides. The former products are therefore chased rapidly into longer products if a suitable substrate is available. The presence of ATP in the caell extracts may explain why the production of dinucleotides is not observed in reactions that involve crude extracts (Fig. 4, lane 2). Most enzymes that retain promoter-binding activity and non-specific catalytic activity also retain the ability to initiate RNA synthesis at t,htr synthetic promoter (Table 2). However, certain of these mutants are unable to initiate specific transcription at a T7 promoter. These include ins640. ins648, ins658 and ins881 (Table 2 and Fig. 5). As the latter enzymes are also unable to synthesize poly(G) on the highly stable poly(dG) . poly(dC) template (see Table 2), we reasoned that some of these mutants might have difficulty in melting int,o the promoter and/or maintaining strand separation during elongation. Tt has previously been shown that. dimethyl sulfoxide (DMSO) can act as a nonspecific denaturant for double-stranded DNA, and that DMSO can enhance initiation by T7 and E. coli RNAP at otherwise weak promoters (Herskovits,

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of T7 RNA

Polymerase

497

around the initiation site. These enzymes were also stimulated by (or dependent upon) DMSO (see above). ins881 RNAP (which is not stimulated by DMSO) was unable to initiate transcription at either type of promoter. (i) Initiation

1 II

I

II

I

II

I

II

I

II

I

II

1

II

Templdte

Figure 6. Initiation of transcription at a promoter that is single-stranded in the initiation region. Panel A, templates were constructed by annealing together 14 and 24-nt oligomers having the sequences shown. NT and T signify non-template and template strands, respectively. Initiation of transcription occurs at position + 1. Panel B, products made from the template indicated were analyzed

as described in Fig. 5.

1962; Nakanishi et al., 1974; McAllister & Carter, 1980). Whereas DMSO had little effect on activity by the w.t. enzyme over the range from 0 to 15% (v/v), the activity of ins640 was dependent upon the presence of DMSO, and appeared to be maximal at a concentration of 15 to 20% (Fig. 5). ins648 was also strongly stimulated by DMSO; ins658 and ins745 were moderately stimulated, and ins881 was unaffected (Fig. 5). (h) Initiation

at a partially

single-stranded

promoter

When T7 RNAP binds to its promoter, the region of the promoter from - 6 to approximately + 2 is melted open and the non-template (NT) strand in this region becomes susceptible to attack by single strand-specific endonucleases (Osterman & Coleman, 1981). The presence of the NT strand in this region is not required for promoter function, and removal of bases from -5 to +6 on this strand has little effect on the initiation of transcription (Milligan et al., 1987; Martin et al., 1988). Since a promoter that is single-stranded in the initiation region comprises, in effect, a “pre-melted promoter”, we were interested in determining the ability of the mutant RNAPs to initiate RNA synthesis at a double-stranded promoter compared with a promoter

that is single-stranded

in the initia-

tion region (Fig. 6). Whereas the w.t. RNAP was able to initiate with approximately equal etlicienties on both forms of the promoter, ins640, ins648, ins658 and ins745 RNAPs were either stimulated by, or dependent upon a single-stranded region

and elongation of RNA at the 410 promoter

synthesis

Although the 24 base-pair promoter fragment described above provides a useful template for initiation assays, a different template is needed to determine the ability of the RNAPs to carry out subsequent steps in the transcription process. The sequence downstream from the 410 promoter in the plasmid pT7-7 has the convenience that, by limiting the availability of substrate, it is possible to assess whether the polymerase can make the transition from an unstable initial transcribing complex (ITC) to a highly processive elongation complex (EC), and whether the RNAPs can carry out the synthesis of 80 nt (or longer) runoff products (see Fig. 7). In reactions involving the detection of these products we have found it necessary to partially purify the RNAPs by chromatography on a column of phosphocellulose. This procedure removes contaminating nucleotides (e.g. ATP) from the preparation, which permits production of the poly(G) ladder. Not all candidate enzymes were amenable to purification in this manner (Table 2), and these RNAPs remain incompletely characterized. . Characterization of mutant RNAPs in this assay system revealed a number of features of interest. ins691 RNAP makes a spectrum of products similar to those made by a proteolytically nicked form of the enzyme (80,000/20,000 M, form) that has been reported by previous investigators to be less processive than intact T7 RNAP (98,000 M, form) and thus, deficient in the synthesis of high molecular weight RNA (Ikeda & Richardson, 1987; Muller et al., 1988). We have found that ins691 and the 80,000/20,000 M, enzyme both exhibit unusual properties during the early stages of transcription, especially under conditions of limiting substrate. For example, whereas the intact enzyme (98,000 M, form) produces low amounts of the 15-nt product at the 410 promoter in the absence of UTP (presumably because the RNAP is arrested in a stable ternary complex), the 80,000/20,000 1M, form and ins691 protein produce significant amounts of this product (see Fig. 7, lane c in 1st 2 panels). We believe that this reflects a lower stability of ins691 and the 80,000/20,000 M, enzyme at this artificial pause site, which causes them to cycle through the reaction, resulting in a greater accumulation of the 15nt

product.

The lower stability of EC involving the SO,OOO/ 20,000 M, and ins691 RNAP is also evident when all four rNTP are present, and is manifest as an increase in the synthesis of products shorter than the expected 80-nt runoff. As the template DNA used in these experiments is not completely cleaved, larger products (> 150 nt) are also produced, and

L. Grosset al.

498 Enzyme WT DMSO _--_

WT

80K/20K - _

BOK/20

ins640

ins646

bcda

bcda

bed

in 745 -

ins745 +,

>80

80

6

r NTPmix

a

ki b c d’k b c d”

a=

GA

bcda

c= GAC

d=

GACU

Figure 7. Initiation and elongation from the 410 promoter. I’roducts made b?- ‘I’7 RNihP from the c#JIOpromoter in t 1~ presence of various mixtures of substrate rNTPs were resolved by electrophoresis in 20% (w/v) polyacrylamide gels. followed bv autoradiography. Substrate mixes contained: a. GTP; b. GTP and ATP; V. GTP, &4TI’ and CTP: d. all 4 rNTPs. r-“PIGTP] was the labeled substrate in all reactions. The 410 promoter initiates with the sequence + I OGGAGA(I(:ACAACGGU. and thus transcription in the presence of substrate mix a is expect,ed tjo give rise to a (: mix b t,o a 6 nt product: mix c to a 15 nt product, and mix d to full-length products. ‘l’hta ladder (by .‘stuttering”), resulting in production of an 80 nt runoff plasmid template (pT7-7) was digested with S’maT prior t)o transoript,ion, product. and also (due to incomplete digestion) in the synthesis of higher molecular weight RXAs t.hat remain at the origin of the gel. The position of the expected RNA products (in nt) are given at the left of the Figure. The enzyme used in each reartion is given above t,he panel; 80K/20K indicates use of proteolytically nicked T7 RX’4P. Where indicated. T)MSO was present, at a concentration of 15”/0 (v/v). are detected as a band at the origin of the gel. Consistent with their decreased processivities, the 80,000/20,000 M, and ins692 enzymes are relatively deficient in the production of these higher molecular weight products. The ins691-encoded enzyme differs from t,he 80,000/20,000 M, enzyme, however, in that it makes a greater abundance of products that appear to be the result of abortive initiation. Furthermore, unlike the situation with the 80,000/20,000 M, enzyme. the presence of ATP (as well as other substrates) does not efficiently suppress the forma-

tion of poly(6) by ins697. The latter observation:: in suggest that’ in.&92 protein may have difliculty making the transit,ion from an ITC t’o an EC’, as well as being less processive. The ins640 and ins648 enzymes are inactive at the $10 promoter in t,he absence of DMSO. and in the presence of this agent the producats made by thr mutant enzymes are different, from those of the w.l. RNAP. Tn the case of ins640, the (i-ladder is shorter than that of the w.t. REAP, and synthesis of poly(G) is not suppressed by the presence of the other rNTPs. Sor is any d&e&able SO-nt product

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Mutagenesis

made under these conditions. Instead, novel transcription products are observed. Although we have not yet characterized these products in detail, we believe that, they may be abortive transcripts 4 and 5 nt in length (GGGA, GGGAG). From these results it appears that ins640 is blocked in the transition from an ITC to an EC. ins648 enzyme is also dependent upon DMSO for activity. However, the G-ladder made by this enzyme is longer than that of ins640, and its formation is partially suppressed by ATP. Like the ins640 enzyme, ins648 RNAP makes abundant quantities of abortive products, but the sizes and relative abundance of these products are different from those of ins640. In particular, the ins648 enzyme accumulates predominantly products in the range of 4 to 5 nt,, whereas the ins640 enzyme makes a somewhat, larger and more diverse set of products. Despite this apparent block at an early stage of transcription, some ins648 enzymes are able to make the transition to an EC, as evidenced by a weak band at 80 nt in the presence of all four rRNPs. ins745 RNAP is not dependent upon DMSO for activity, but is stimulated by this agent. In the absence of DMSO, very little production of poly(G) or abortive initiation products is observed. Despite this apparent deficiency in initiation, ins745 protein is able to initiate RNA synthesis and to produce full length runoff products in the absence of DMSO. Under these conditions, the mutant enzyme appears to be highly processive, as judged by the absence of products 80 nt. The addition of DMSO restores the ability of ins745 enzyme to produce the G-ladder and the 6-nt product, and also stimulates production of full-length products. ( j) Recognition

of termination

Polymerase

I4 Ik I I IO 9 8 7 6 5 4 3

2

signals

In related work we have determined that T7 RNAP recognizes three distinct types of transcription stop signals: the termination signal that occurs naturally in T7 DNA (T4), a stretch of 40 A residues in the template strand (which would direct the incorporation of a U,,-tract), and a poorly characterized signal present in the human parathyroid hormone (PTH) gene (L. MacDonald & W. T. McAllister, unpublished results; Mead et al., 1986). None of the mutants that we tested exhibited altered termination properties at these signals (Table 2). (k) ins881 mimics a previously “foot” mutant

of T7 RNA

12345 Figure 8. Initiation of ins881 requires high concentrations of enzyme and GTP. Products made by w.t. RNAP (lane 1) or ins881 RNAP (lanes 2 t’o 5) at the $10 promoter in the presence of GTP as the sole substrate were analyzed as described in Fig. 7. The concentration of GTP was either @5 mM (lanes 1 to 3) or 5.0 rnM (lanes 4 and 5). Enzyme concentration was either 10 ng (lanes 1, 2, 4) or 100 ng (lanes 3 and 5) per reaction. The sizes of the products in the G-ladder (in nt) arc indicat,ed to the left.

characterized

Previous investigators have demonstrated that T7 R’NAPs that lack the C-terminal Phe and Ala residues are less processive and are prone to abortive initiation. These RNAPs can be created either by mutation (“foot” mutant) or by removal of the terminal residues by carboxypeptidase (Mookhtiar et al., 1991). The ins881 protein contains an insertion of two amino acid residues at position 881, and

although the terminal Phe and Ala residues are retained in this polymerase, they are expected to be displaced as a consequence of the insertion. We were therefore interested in determining whether the ins881 enzyme exhibited properties similar to that of the “foot” mutant. As shown in Figure 8, ins881 is inactive on the 410 promoter under normal conditions. However, as previously reported for the

500

L. Gross et al.

“foot” mutant, the ability of ins881 to synthesize a (truncated) G-ladder can be detected by combination of a tenfold increase in polymerase concentration and a tenfold increase in the GTP concentration.

4. Discussion (a) Method of mutagenesis The method of mutagenesis that we have chosen results in the introduction of six base-pairs into the RNAP gene at positions that are defined by the choice of restriction endonuclease. Whereas insertion between codons results in the introduction of two amino acid residues into the protein, with no other changes, insertion within a codon has the potential to alter the residues that flank the site of insertion. In the mutants characterized in this study, 19 of 34 have a single amino acid residue substitution in addition to the insertion (Table 1). In agreement with previous observations (Zebala & Harany, 1991), the insertion of six base-pairs-seems to result mostly in localized alterations in protein structure, as 25 out of 34 RNAP mutants are active in vivo or exhibit partial enzymatic activity in vitro. Mutant enzymes with partial enzymatic capacities are of particular interest, as they may be disrupted in one function but not another, potentially allowing assignment of the affected function to a particular region of the RNAP. (b) Comparison of T7 RNA polymerase other phage-like RNA polymerases

with

T7 RNAP is the prototype of a class of related DNA-dependent RNAPs having a single subunit. These include RNAPs from other phages such as T3, SP6, Kll, ghl and BA14 (McGraw et al., 1985; Butler & Chamberlin, 1982; Kotani et al., 1987; Dietz et al., 1990; Towle et al., 1975; Korsten et al., 1979; Mertens et al., 1982). The homology of this class of enzymes to RNAPs of mitochondria and to RNAPs encoded by mitochondrial plasmids has also been noted (Masters et al., 1987; Levings & Sederoff, 1983; Oeser & Tudzynski, 1989; Robinson et al., 1991; Chan et aE., 1991). It is significant that mutations that lead to complete inactivation of T7 RNAP lie predominantly in regions where the amino acid sequences are highly conserved. (c) Consideration (i) ins691 protein to complement

of individual

is transcriptionally T7 gene 1~ phage

phenotypes active but fails

Although the ins691 enzyme is active in vivo, as assessed by moderate levels of chloramphenicol resistance in BL21(DCAT4) cells, it is unable to complement the growth of T7 gene I- phage. The defect in support of phage replication is not due simply to a lower capacity for RNA synthesis, as other mutants that exhibit lower levels of chloramphenicol resistance are able to complement the

defective phage. As noted above, T7 RNAP plays multiple roles in phage replication, and some of these may involve specific protein-protein interactions that are independent of the enzyme’s basic function in RNA synthesis. ins691 protein may be defective in one (or more) of these interactions. The mutation in ins691 lies in a region of T7 gene I where other mutations that affect the interaction of T7 RNAP with phage lysozyme have been mapped (Moffat & Studier, 1987; X. Zhang & F. W. personal communication). Lysozyme Studier, inhibits the phage RNAP at late times after infection (Moffat & Studier, 1987) and RNAP mutants that exhibit increased sensitivity to lysozyme (poZisL) have been isolated (X. Zhang & F. W. Studier, personal communication). Although polIsL mutants are unable to support the replication of TS gene 7 phage, they do support replication of phage that are also defective in the gene for T7 lysozyme (gene ,Y,“). However. ins691 is unable to complement growth of T7 gene 7-, gene ??*5 phage, and therefore does not appear to be a ~01”~ mutant (X. Zhang & F. W. Studier. personal communication). An alternative explanation for this phenomenon may be that the ins691 enzyme is able to transcribe chromosomal DNA but unable to transcribe phage DNA, perhaps due to a different, state of the template (e.g. supercoiling, or complexing with other proteins). In this regard, it is important to note that irls69l prot,ein exhibits unusual properties in vitro. In particular, the mutant enzyme appears to be prone to abortive initiation and to premature termination. Tts lower processivity gives rise to a pattern of products similar to that observed for the proteolytically nicked enzyme (Fig. 7). Perhaps the chloramphenicol-resistance gene is shorter than some of the essential T7 transcription regions. or has fewer pause sites. (ii) 7’he mutation ill ins745 lies within promoter-binding sitr

or near a

The mutation in ins745 lies very close t,o N748. a residue that, we have previously shown to he involved in descrimination of base-pairs in the - 11 region of the promoter. (Joho et al.; 1990; Raskin et al.: 1992), and the propert,ies of the ins745 enzyme are consistent with disruptions in a promoterbinding site. ln particular, this RNAP exhibits weaker promoter binding than the w.t. enzyme (our unpublished results), and a diminished ability to transcribe double-stranded DNA (poly(dG) . poly(dC)) as opposed to single-stranded DNA (poly(dC)). Initiation at, a synthetic promoter is weak. but is stimulated by DMSO, and t*he mutant singleenzyme prefers t)o initiate at a partially stranded promoter as opposed to one that is completely double-stranded. We have previously suggested that this region ot T7 RNAP might not only be involved in sequencespecific promoter recognition, but also might provide contacts that are important for torsional constraint in the induction of structural alterations, such as promoter melting (Joho et al.. 1990). The

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Insertion

Mutagenesis

preference of ins745 RNAP for a partially singlestranded promoter may reflect a weakened ability to bind to the promoter, and/or to form an open complex. Alternatively, recognition of the promoter may involve an “induced fit”, and DMSO, by lowering the stability of the DNA helix, may allow the transition to the optimal binding configuration at a lower energy cost. ins745 RNAP exhibits unusual properties in the early stages of initiation at the 410 promoter (Fig. 7). In the absence of DMSO very little “stuttering” (,production of a G-ladder) occurs, and low amounts of the 6-nt product are found in the presence of GTP and ATP. A possible explanation for this observation is that “stuttering” and the repeated production of abortive initiation products may require a more stable association of the enzyme with the promoter than does initiation (and promoter clearance). The ins745 mutation may lower promoter affinity sufficiently to affect the former process to a greater extent than the latter. (iii) ins159 enzyme exhibits normal catalytic activity but is blocked in promoter binding; this region of the T7 REAP exhibits homology to the E. coli sigma factor ins159 RNAP exhibits high levels of non-specific catalytic! activity on poly(dC) and poly(d1). poly(dC), but weak activity on poly(dG) . poly(dC). It also exhibits extremely weak promoter binding and weak initiation at a synthetic promoter. Its activity is not stimulated by DMSO nor at a singlestranded promoter, suggesting a defect at the level of binding and not with melting or initiation (as for ins745). Consistent with this, the ins159 RNAP gives a weak but normal pattern of products at the $10 promoter (not shown). The k-259 mutation lies immediately adjacent to a region of the phage RNAP that exhibits a striking homology to region 2.4 of the E. coli sigma factor (Fig. 9). This region of the sigma factor is thought to be involved in specific binding to the E. coli promoter, and is believed to interact with bases in the -10 region of the promoter (Helman & Chamberlin, 1988; Daniels et al., 1990; Waldburger et aZ., 1990; Siegele et al., 1989). Another T7 RNAP mutation that lies within the region of homology causes a complete inactivation of the RNAP (insZ44; Table 2). (iv) ins559, ins640 and ins648 lie in a region of T7 RNAP that is homologous to the active site of DNA polymerase, and exhibit properties that would be expected for active site mutants Delarue et al. (1990) have noted a homology in three sequence motifs found in many DNA polymerases and single-subunit DNA-dependent RNA polvmerases. Two of these motifs (motifs A and C; see Fig. 10) are also found in RNA-dependent RNA polymerases as well as RNA-dependent DNA polymerases. In E. coli DNA polymerase I (pal I) these three motifs are located near the active site, suggesting that these enzymes may have evolved

of T7 RNA

Polymerase

501

A.

0'0 TmNAP