Eur. J. Biochem. 195,841 -847 (1991) 0FEBS 1991 001429569100101K

Lys631 residue in the active site of the bacteriophage T7 RNA polymerase Affinity labeling and site-directed mutagenesis Tatyana G. MAKSIMOVA’, Arkady A. MUSTAYEV’, Evgeny F. ZAYCHIKOV’, Dmitry L. LYAKHOV’, Vera L. TUNITSKAYA’, Akhror Kh. AKBAROV’, Sergei V. LUCHIN’, Vladimir 0. RECHINSKY ’, Boris K. CHERNOV’ and Sergei N. KOCHETKOV’

’ Limnological Institute, Siberian Division of the USSR Academy of Sciences, Irkutsk, USSR V. A. Engelhardt Institute of Molecular Biology, USSR Academy of Sciences, Moscow, USSR (Received September 27,1990)

-

EJB YO 1154

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [a32P]UTPwas carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys63 1 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys + Gly mutant enzyme, anomalous template binding was observed. Bacteriophage T7 DNA-dependent RNA polymerase (T7RP) is known to be one of the simplest enzymes catalyzing RNA synthesis. It is a single polypeptide chain composed of 883 amino acid residues [l, 21. The primary structure of the enzyme has been determined recently [3, 41. Little is known about the functional groups and the topography of its active site. Three main approaches are used to study the active sites of enzymes: (a) X-ray analysis of enzyme-substrate complexes; (b) affinity labeling of an enzyme with substrate analogs; (c) site-directed mutagenesis of the amino acid residues in the active site and functional analysis of mutant proteins. X-ray studies of T7RP are in the rudimentary stage; the crystallisation of the enzyme has just been reported [5]. At present therefore the topography of the T7RP active site can be investigated using a combination of approaches (b) and (c), i.e. the identification of the functionally important amino acid residues by affinity labeling and subsequent studies of their role using a site-directed mutagenesis.

other reagents were obtained either from Serva or from Boehringer Mannheim.

Enzymes T7RP (150000 Ujmg) wild type was isolated from Escherichia coli containing the expression plasmid pACT7 (Fig. 1 B). This vector includes an entire coding sequence for a thermosensitive CIrepressor and a promoter-operator region ORPR of bacteriophage 2 as well as a T7RP structural gene. The wild type of T7RP as well as its mutant forms were purified as in our previous work [6]. DNA polymerase, T4 DNA ligase and restriction endonucleases were obtained from Amersham and used according to the manufacturer’s instructions. Bacterial strains, bacteriophages and plasmids

These are listed in Table 1. C1a1

EcoRI

MATERIALS AND METHODS

Clal

Materials

Ribonucleoside and deoxyribonucleoside triphosphates were purchased from Serva, radiochemicals and M13 sequencing kit from Amersham, Zeta-Probe blotting membranes from Bio-Rad, low-melting-point agarose from Sigma and agar, yeast extract and bactotryptone from Difco. All Correspondence to S. N. Kochetkov, V. A. Engelhardt Institute of Molecular Biology, USSR Academy of Sciences, Vavilova st. 32, 117984 Moscow, USSR Abbreviations. T7RP, bacteriophage T7 DNA-dependent RNA polymerase; Pr, 410 promoter-containing fragment of pGEM-2. Enzymes. Bacteriophage T7 RNA polymerase (EC 2.7.7.6); restriction endonucleases (EC 3.1.22.4); DNA polymerase (Klenow fragment) (EC 2.7.7.7); T4 DNA ligase (EC 6.5.1.1); trypsin (EC 3.4.21.4).

?I

EC~RI

A

B

Fig. 1. Structure of theplasmidspAC1 ( A ) andpACT7 (B). pACl is the derivative of pACYC184 [I21 containing the coding sequence for CI repressor and ORPRregion of bacteriophage A. pACT7 is derived from pACl by inserting the structural gene of T7RP as a 2.7-kb BamHI - BamHI fragment

842 Table 1. Bucteviul struins, p h u p s and plasmids used in the work An asterisk in the final column indicates that the strain was isolated in this work; w. t. = wild-type Organism

Strain

Genotype

Reference

E coli

RZ1032 SMlOl MI 3mp18 MI 3mpl8T7 jc1857 pAR1219 pACYClS4 PAC 1 pACT7 pKG31G pKG31L pKG31 R

Hfr KL16 POI45 [lysA/61-61)],d u t l , ungl, thil, relA1lZhd-279 :: Tn 10. supE44 A(1ac-pro), supE, t h i l / F ( p r o A B, traD36, lacIq, lucZAM15)

PI

Bacteriophages Plasmids

T7RP clts A p R ; lucLJV5-T7RP CmR; Tet‘ CmR; TetS;C I 8 5 7 - 0 ~ p ~ CmR; Te?’; C I ~ ~ ~ - O R P R -(W.t.) T~RP Cm’; Tets; c1857-ORPR-T7RP(Lys631 + Gly) CmR;Tets; C I ~ ~ ~ - O R P R -(Ly~631 T ~ R P +Leu) CWIR; Tets; C I ~ ~ ~ - O R P R -(Lys631 T ~ R P --t Arg)

Assuys T7RP activity was assayed as in [6]. The incubation mixture (25 pl) contained the following components: 50 mM Tris/ HCI pH 7.8, 20 mM MgC12, 10 mM 2-mercaptoethanol, 0.4 mM (each) ATP, UTP, CTP and GTP; 3 x 10’ cpm [a32P]ATP;20 - 40 pg/ml pGEM-2 (Promega Biochem); 0.1 0.2 pg T7RP (wild type or mutants). The apparent K, and k,,, values ( & SE) were determined at the saturated concentrations of nonvariable substrates using the method of Wilkinson [7]. The affinity labeling experiments were performed using a 60-bp double-stranded synthetic oligonucleotide containing the consensus sequence of T7 class 111promoters with a single substitution

(:2 --f

. in

+ 2 position of the transcribing region

~31. A full-length EcoR1-restricted pGEM-2 or a 106-bp promoter-containing fragment (Pr) were obtained as in [6]. The binding of the enzyme to promoter was carried out on nitrocellulose filters (Millipore HAWP 0.45 pm). The incubation mixture (10 pl) contained the following components: 14 mM Tris/ HCI pH 7.9, I3 mM NaCl, 5 mM MgCI2, 0.5 pmol T7RP (wild type or mutants). [32P]lpGEM-2was used as indicated in the Results. [32P]Pr(106 bp) was used in the same concentrations. The samples were incubated at 37°C for 5 min, applied to nitrocellulose filters, washed with buffer, dried and the radioactivity was measured with a toluene-based scintillator.

[91 [91

*

ume of 2 M hydroxylamine in 0.2 M K 2 C 0 3 (pH 10.0) was added and the mixture was incubated at 37 ’C. The aliquots of the reaction mixture were analyzed by electrophoresis and autoradiography. Cleavage of the labeled,fragment C with N-chlorosuccinimide, bromine and cyanogen bromide

Fragment C (see Results) was isolated by electrophoresis after the affinity labeling of T7RP followed by cleavage with hydroxylamine. The fragment was eluted from the gel and treated at 25°C with either 10 mM N-chlorosuccinimide in 0.2 M ammonium formate (pH 4.0), or 1 mM bromine in the same buffer, or 100 mM cyanogen bromide in 0.05 M HCl for 15 min. The reactions were stopped with 2-mercaptoethanol (up to 1%) and triethanolamine (pH 8.5, up to 0.1 5 M), heated at 56 “C for 15 min and analyzed as described above. Recombinant D N A techniques

Plasmid DNA and replicative M13 DNA were isolated from E. coli according to Birnboim and Doly [18]. Singlestranded M13 DNA was prepared as described in [19]. Transformations with plasmid DNA, transfections with MI 3 and selection of M I 3 recombinants were carried out by the standard procedures [20, 211. To verify the size and orientation of an insert, the recombinants of plasmids and M13 were screened according to [IX].

Gel electrophoresis,

Subcloning of T7RP gene

Polyacrylamide gel (109’0) electrophoresis was carried out according to Laemmli [14]. Gels containing 32P-labeled samples were autoradiographed with an RMV film.

The sequence encoding T7RP has been excised from plasmid pARI219 [ l l ] by partial digestion with Sau3A and recloned into the BamHI site of M13mp18. The resulting M13mp3 8T7- provided recombinant phages containing the sequence complementary to the coding one of the original gene. The presence of two BamHI sites flanking the cloned sequence in M13mpIST7- was confirmed by restriction mapping.

Affinity labeling oj 7‘7RP

The affinity reagent, o-formylphenyl ester of GMP (o-PhGMP), was synthesized as in [15]. T7RP was labeled with oP h- GMP using [ X - ~ ~ P I U as T Pa radioactive substrate as in [16, 171. Cleavage qf the labeled T7RP with hydroxylamine

Labeled T7RP was denatured with 1% SDS in the presence of 1 O/O 2-mercaptoethanol at 37 “C for 30 min. An equal vol-

Oligonucleotide printers for site-directed nzutagenesi.5

The mutagenic primers (the asterisks mark mismatched bases) 5’-ACTCGCAGTGTGACTG * G * C * CGTTCAGTCATGACG-3’ (K631G), 5’-ACTCGCAGTGTGACTC * T * G * CGTTCAGTCATGACG-3’ (K631L) and 5’-ACT-

843 CGCAGTGTGACTC * G * C * CGTTCAGTCATGACG-3’ (K631R) were designed from the published nucleotide sequence of T7RP [3,4] and synthesized according to [22].

trypsin 1

7

588-58;

289-290

Met

Asn-Gly-Asn-GI B

A

A,y3 C

A

hydroxylamine

RESULTS Affinity labeling of T7RP

A

The affinity label, o-formylphenyl ester of GMP (o-PhGMP) [15] was used to modify the enzyme.

A + B

7 B * C

0

t C

@0-;;0-(5)Guo

1

2

3

CHO The aldehyde group of this analog is in a close proximity to its phosphate residue. Such proximity makes the formation of a Schiff base between this aldehyde group and the &-amino group of lysine(s) interacting with the phosphates of initiating NTP in the active site of the enzyme highly probable. T7RP was modified according to Scheme (I), which allows one to label the active-site region with high selectivity.

-590

a.a.

B

-290

a.a.

E-NHZ-tOHC-R-pG +E-N

=

CH-R-pG-

NaBH4

(1)

E - NH -CH2 -R-pG E - NH - CH2 - RpGp*U where E = T7RP; OHC-R-pG = o-Ph-GMP and PPP*U= [LX-~~PIUTP. A synthetic ds DNA fragment (60 bp) containing the consensus sequence of T7 class I11 promoters was used as a template. A single change

if:+I)

was introduced in the

Fig. 2. Hydroxylamine treatment of affinity labeled T7RP. (A) Schematic representation of the T7RP cleavage. The sites of cleavage as well as the label position ( 0 )are shown. (B) Electrophoretic patterns of the products of the hydroxyamine cleavage (10% PAGE). Lanes 1, 2, 3, 2-h, 4-h and 9-h incubation, respectively; a.a. = amino acid residues

+2

position of the transcribing region. Such a change did not affect the enzyme binding, but allowed us to stop the elongation of the nascent RNA chain after the addition of the second nucleotide (pU) and made it easier to localize the affinity label. The localization of the ajjinity labeled amino acid residue

To localize the affinity label we used the method of cleaving the labeled protein statistically only once per molecule [15,23, 241. Hydroxylamine is known to cleave the Asn-Gly peptide bonds [25] at pH 9 - 10. There are two such bonds in a T7RP molecule, namely, Asn289-Gly290 and Asn588-Gly589. Thus the cleavage by hydroxylamine under the conditions mentioned above should yield fragments composed of 594, 588, 294 and 289 amino acid residues. As can be seen from Fig. 2, two fragments consisting of approximately 590 and 290 amino acids contain the 32P label. The radioactivity in the smaller fragment shows that the terminal third of the enzyme (fragments A or C, Fig. 2) has been modified. The limited digestion of the labeled T7RP with trypsin (which is known to split the bond Lys172-Arg173 [26]) prior to hydroxylamine treatment does not change the mobilities of the labeled fragments. Therefore the affinity label seems to reside in the C-terminal third of the enzyme (fragment C in Fig. 2).

Further studies are illustrated in Fig. 3. For precise localization of the affinity label, the fragment C was purified by preparative electrophoresis and subjected to limited cleavage at Trp, Tyr and Met residues. The treatment of the labeled fragment with N-chlorosuccinimide, which is known to cleave polypeptide chains at Trp residues, yielded an electrophoretic pattern corresponding to that expected for N-terminal peptides (Fig. 3). Hence the shortest labeled peptide, containing approximately 100 amino acid residues, corresponds to the N-terminal part of fragment C and apparently is formed once the polypeptide chain has been split at Trp682. The N-terminal fragment of 31 amino acids (corresponding to cleavage at Trp620) is absent, but there is a C-terminal radioactive fragment of 263 amino acids which is formed upon the cleavage at this residue. Thus the affinity label is located between Trp620 and Trp682. As the nature of the reactive group (formylphenyl) determines the attachment only to an &-aminogroup of Lys via formation of a Shiff base, three lysine residues (631, 642 and 663) may be possible targets for modification. After the cleavage of fragment C with bromine (at Trp and Tyr residues) and cyanogen bromide (at Met residues), we finally localized the position of the affinity label. The electrophoretic patterns for the products of these cleavages corresponded to the theo-

844 A

635

Met

666 677 696 682

620

TrP

750

698

727736

1

632861 797

589

G~Y

Ala &631

676

739

802846

LYS

C

B

N-terminal 1

846836, 802-

797'

739, 733, 727/

698, 682676-

639920-

2

C-terminal 1

3

2

3

I1 620676682'

698'

736, 739-

666 802*

635 - 666 77 - 6696 - 750 -832

846'

-861

Fig. 3. Localization of the aflinitl; label on the products of the cleavage of fragment C. (A) Arrangement of Trp, Tyr and Met residucs on the polypeptide chain of the fragment C. (B) Theoretical electrophoretic patterns designed on the assumption that the label is at the N-terminus (left) or C-terminus (right) of fragment C. Cleavage with bromine (lane I), N-chlorosuccinimide (lane 2) and BrCN (lane 3). (C) Electrophoretic patterns of the cleavage of the fragment C by bromine (lane I), N-chlorosuccinimide (lane 2) and BrCN (lane 3). Lane 4 represents untreated fragment C

retical distribution of N-terminal peptides and differed from that of C-terminal ones. In both cases radioactive peptides of about 50 residues were present. These were apparently products of single-hit cleavage at Tyr639 and Met635. This means that the affinity label is situated to the left of Tyr639 and Met635 and resides at Lys631. Site-directed mutagenesis and expression of mutant genes of T7RP

Site-directed mutagenesis was employed to investigate the role of the Lys631 residue in the function of T7RP. Lys631 was substituted by Arg, Gly or Leu by the method of Kunkel [8]. The template for mutagenesis was isolated from MI 3mp18T7- phages produced after transfection of E. coli RZ1032 (dut-, ung-). The E. coli strain JMlOl (dut', ung') was transformed after the primer phosphorylation, annealing extension and ligation. Plaques containing the mutant phages were identified by hybridization with 32P-end-labeledmutagenic oligonucleotides [27]. The mutations were verified by dideoxy-sequencing [28]. To express T7RP mutant forms, the corresponding genes were excised from the MI 3mp18T7- replicative forms as

BamHI - BamHI fragments and recloned into the expression vector pACl (Fig. l), generating pKG31G (Lys631+ Gly), pKG31L (Lys631 +Leu) or pKG31R (Lys631 4 A r g ) .

Kinetic studies of R N A polymerase mutants

The mutant enzymes G (Lys631 + Gly), L (Lys631 +Leu) or R (Lys631+ Arg) were purified to homogeneity and used in kinetic experiments. The decreased stability in the course of storage (- 20 "C, 50% glycerol) was observed for all three mutant proteins. The half-life of the activity was 7 - 10 days for R and 2 - 3 days for G and L enzymes. The inactivated enzymes, however, retained the ability to bind a DNA template. The RNA polymerase activity of freshly prepared mutant enzymes (as compared to the wild-type polymerase) appeared to be 15-20% for R, 3-5% for G and 1-1.5°/~ for L. The activity of the mutant enzymes was expressed mainly as a decrease of turnover (k,,, values; Table 2). In the case of G, K , values for all the four NTPs were close to those of the wild-type enzyme. For R and L, KkTpand KZTPwere 3 - 5 times higher.

845 0.5

1

3

L

nn

0

A

40

20

60

80

100

120

140

[pGEM-21, nM

z

5

. 4

’D

2 3

Q c\1

z2 0

0

20

40

60

80

I00

120

140

[pGEM-2) nM

Fig. 4. Binding of the mutant T7RP enzymes with promoter-containing DNA. Curve 1, wild type; curve 2, L (Lys631 -+Leu); curve 3, R (Lys631 +Arg); curve 4, G (Lys631+ Gly). For details see Materials and Methods. Note the different vertical scales in A and B

Table 2. Kinetic characteristics of mutant T7RP enzymes Apparent Kmand k,,, values were determined as described in Methods. SE for these parameters were within the limits of 10- 15% Mutation

kc,,

K:rp

S-1

PM

230.0 Wild type G (Lys631 -+Gly) 12.5 8.0 L (Lys631 -+Leu) R (Lys631 +Arg) 62.7

55 39 154 149

CTP

KkTP

KZTP

50 80 76 60

36 48 138 I40

143 170 200 170

The binding of R and L to full-length linearized pGEM-2 is close to that of the wild-type enzyme (Fig. 4A) while the binding of G increases (Fig. 4B). The use of the short 106-bp promoter-containing fragment of pGEM-2 (Pr) shows the same effect (data not shown). The”anomalous1y high binding of G is apparently due to an unspecific DNA-protein interaction: the promoter-containing DNA is readily displaced from the complex by unspecific DNA. At a 100-fold excess of the latter, the binding of G is close to that of all other T7RP types (Fig. 5). DISCUSSION Up to now, little has been known about the topography of the active sites of bacteriophage RNA polymerases. The

method developed by us has made it possible to map amino acid residues participating in the binding of the initiating nucleoside triphosphate in RNA polymerases of various types [15, 23, 24, 34-36]. In this study we modified T7RP with ofPh-GMP and identified the Lys631 residue as a target for this affinity label. Lys631 is located in the polypeptide region which is identical to that of T3 RNA polymerase and is highly similar to sequences found in related enzymes from phages K11 and SP6 and yeast mitochondria [30 - 331 (Fig. 6). This fact indicates that structures comprising the active sites of these enzymes are evolutionarily stable. Lysine and arginine residues are often found in the active sites of nucleotide-binding proteins, particularly, of bacterial, archeabacterial and eucariotic RNA polymerases [15, 23, 24, 341. Apparently their role is an ionic interaction with the phosphate groups of substrates. Taking the structure of the affinity label (o-fPh-GMP) into account, it would be reasonable to suppose that Lys631 participates in the interaction with the a-phosphate of the initiating nucleoside triphosphate. In order to gain further information about the role of Lys631, we replaced this residue with Gly, Leu or Arg. The selection of mutations was based on the following considerations. a) When Lys is substituted by Gly, there is no side chain in position 631 and thus this residue can not interact with the phosphate group of the substrate. b) When Lys is substituted by Leu, there is no electrostatic charge in position 631 but the bulky hydrophobic substituent is retained here.

846 05

0' 0

I

20

40

60

80

I

I

100

120

unsp.DNA/pGEM-2

Fig. 5. The di,splcicemcvit ofprotnofer,fiom the complex with T7RP by unspecific D N A . Curve 1, wild-type T7RP; curve 2, G-mutant form of T7RP

T7IT3

iK6311

IT R S V TKR S V M T L A Y

K11

(~6%)

In

T R K V TKR S V M T

G

I

LA Y G

Fig. 6. Similurity of the region of the affinity labeling of T7RP (Lys631) with the sequences qf the R N A polymeruses of phuges T3 [30/, K l l [31], SP6 [32] and the enzyme from yeast mitochondria [33/. Identical sequences are boxed

c) When Lys is substituted by Arg, this region still has the same net electrostatic charge but the steric complimentarity between the interacting groups may be affected. The mutant enzymes were obtained in high yield and used in kinetic experiments. The observed decrease of R, G and L activities may be interpreted as follows. Firstly, their partial inactivation can occur due to incorrect folding of the polypeptide chain as a result of mutation. Secondly, this effect may be attributed to alteration or elimination of the specific function of the mutated amino acid residue. In the absence of Xray data, it is impossible to evaluate directly how the threedimensional structure of the mutant protein fits in with that of the wild-type T7RP. The relatively small changes of K , are indirect evidence in favour of at least partial preservation of the structure of the mutant proteins as coinpared with that of the native enzyme. The secondary structure analysis by the method of Garnier [29] (data not included) has shown that Lys631 is located in the middle of the sequence (622- 638) which forms a p-structure stretch. The mutations Lys 4 Arg and Lys 4 Leu should not cause dramatic changes in the secondary structure of this region while the Lys-Gly change can break the j-conformation at the site of the mutation. There are relatively small but reliable differences between the kinetic properties of the mutated enzymes. For instance, R has a lower affinity for ATP and UTP than L, G and the wild-type T7RP, whereas the affinity for GTP and CTP is close for all the four enzymes. The anomalously high binding to unspecific DNA is a peculiar feature of G. In this connec-

tion one may suggest that Lys631 mutations associated with local rearrangements in the active site can cause changes in the entire T7RP mechanism. It must be mentioned that a similar studies on the active site of E. coli RNA polymerase have been recently published [37]. It was shown that a single change of Lys residue in the vicinity of the active site causes the pronounced effect on the ability of the enzyme to synthesize products longer than dinucleotide and its escape to the elongation step. In any case, more information is needed to elucidate the functional role of Lys631. Further experiments on srte-directed mutagenesis, as well as kinetic investigations, are now in progress.

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