Deletion Analysis of Qbeta Replicase
PARTICIPATION OF THE CARBOXYL-TERMINAL REGION OF THE beta -SUBUNIT PROTEIN IN TEMPLATE RECOGNITION*

(Received for publication, October 23, 1996, and in revised form, March 19, 1997)

Yoshio Inokuchi Dagger and Masayuki Kajitani

From the Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya-shi, Tochigi 320, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have analyzed one of the functional domains of Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta . Deletion mapping analysis of the carboxyl-terminal region of the beta -subunit protein revealed that the terminal 18 amino acid residues (positions 571-588) are dispensable for the replicase reaction. Subsequent deletions up to the Ala-565 residue reduced the RNA polymerizing activity of the replicase in vivo but increased it in vitro. The mutant replicases with enhanced in vitro RNA polymerizing activity were found to have relaxed template specificity for ribosomal RNAs and cellular RNAs as well as Qbeta RNA. Deletions beyond the Ile-564 residue abolished both the RNA polymerizing activity and the binding ability to midivariant (MDV)-poly(+) RNA, a derivative of a natural template for Qbeta replicase, MDV-1 RNA. These results suggest that the carboxyl-terminal part of the beta -subunit participates in RNA recognition of Qbeta replicase.


INTRODUCTION

With the development of modern gene technology, it became feasible to analyze the relationships between the structures and functions of gene products. In the case of RNA coliphages, the complete nucleotide sequences of representative phage genomes were determined (1-4), and analysis of the structure of each gene also became possible. In attempts to clarify the functional domain of the polymerases, several polymerase genes have been extensively characterized.

An RNA-dependent RNA polymerase (RNA replicase) of RNA coliphage Qbeta consists of four subunits. Three of them are host-derived proteins (ribosomal protein S1, and protein elongation factors, EF-Tu and EF-Ts) (5). Only the beta -subunit, which is composed of 588 amino acid residues, is encoded by phage RNA. The central region of the beta -subunit is well conserved in RNA coliphages, and this region is thought to contain common structural features for such as the assembly of the subunits and catalysis for RNA synthesis (6). A sequence motif, Gly-Asp-Asp, which has been identified in RNA-dependent RNA polymerases of many viruses and is considered to be the active site for polymerization (7, 8), is located in the central part of the beta -sububit protein (9). Recently, this motif sequence of the Qbeta and polio virus 3Dpol proteins was found to take part in the binding of metal ions necessary for RNA polymerization (10, 11).

The Qbeta replicase can specifically transcribe in vitro the Qbeta RNA as well as the RNA from closely related phages (5). S1 and another host protein, HF-I (12), are required for the recognition of only Qbeta viral plus-strand RNA as a template. In contrast, Qbeta replicase lacking S1 and HF-I transcribes poly(rC), MDV-1 RNA, and Qbeta minus-strand RNA, as does the holoenzyme of Qbeta replicase (5). RNA replicases from phages MS2, GA, and SP share the host subunits of Qbeta replicase except for HF-I, but they have the different template specificity. Therefore, it is reasonable to think that some factor(s) determining the enzyme specificity must be present in the beta -subunit.

The beta -subunit proteins of MS2 and GA replicases are approximately 10% shorter than those of Qbeta and SP replicases, with most of the extra amino acids being located in the carboxyl-terminal region (4). Furthermore, the carboxyl termini of the beta -subunit are more divergent in sequence (Fig. 1). It is therefore likely that this region may contribute to template discrimination.


Fig. 1. Amino acid sequence similarity of the carboxyl-terminal region of the beta -subunit protein among MS2, GA, Qbeta , and SP replicase. Each amino acid is represented as a single letter. Numbers start from the amino-terminal end of the beta -subunit protein. Asterisks indicate identical amino acids. Bars are used to represent probable insertion or deletion sites. Sequence data are taken from the Refs. 1-4.
[View Larger Version of this Image (41K GIF file)]

To clarify in detail the physiological role of the carboxyl-terminal part of the beta -subunit in the Qbeta replicase reaction, we constructed a series of plasmids harboring the beta -subunit gene with deletions at its 3'-terminal end and examined the effects of the deletions on the enzyme functions. The carboxyl-terminal 18 amino acid residues were found to be dispensable for the replicase reaction, but a larger deletion made the enzyme defective.


EXPERIMENTAL PROCEDURES

Bacteria, Phage, and Plasmids

Escherichia coli A/lambda (sup pro/F+) and BE110 (sup+ rel-1 T2r tonA22 phoA4 Hfr) were used as indicators. E. coli 594/F' lacIq (sup+ gal Strr recA44/F'lacIq lac+ proAB) was used as a recipient for transformation and for growing phages. Phage Qbeta sus51 was described previously (13). Plasmid pRQ1, which contains the wild-type beta -subunit gene of Qbeta replicase, was used to construct plasmids carrying various sizes of the replicase gene lacking the 3'-terminal region. Plasmid pUC-MDV-LR carrying the segment corresponding to MDV-poly(+) RNA was described previously (10). MDV-poly(+) RNA is 244 nucleotides long and is a derivative of naturally occurring MDV-1(+) RNA (14), for which Qbeta replicase exhibits strong template activity. Plasmid ZpQbeta -32 (15) was provided by C. Weissmann.

Materials

Poly(rC) and DNase I were purchased from Pharmacia Biotech Inc. [gamma -32P]ATP, [alpha -32P]dCTP, [alpha -32P]GTP, and [alpha -32P]UTP were obtained from Amersham Corp. Restriction endonucleases were purchased from TaKaRa Shuzo. AmpliTaq DNA polymerase was from Perkin-Elmer. The 16 and 23 S ribosomal RNAs of E. coli MRE600 were from Boehringer Mannheim.

Construction of Plasmids Carrying the beta -Subunit Gene

Plasmids carrying the beta -subunit gene, of which the 3'-terminal end was shortened to various extents, were constructed using the polymerase chain reaction method. Oligonucleotide RQ1-f2 has a nucleotide sequence corresponding to positions 3328-3351 of the Qbeta cDNA sequence including the cleavage site for the restriction enzyme, XhoI, and was used as a forward primer (Table I). The oligonucleotides listed in Table I were used as reverse primers, containing an artificial stop signal leading to premature termination of beta -subunit protein synthesis and having the site for BglII for inserting the DNA fragment into the vector plasmid. Amplification of the DNA fragments was carried out according to the manufacturer's instructions. The amplified DNA fragments were digested with XhoI and BglII and then ligated to plasmid pVAR-AD6 DNA (9), which harbors the beta -subunit gene lacking the 3'-terminal part and was previously cleaved with XhoI and BamHI. The nucleotide sequences of the amplified DNA regions were analyzed by the chain termination method (16) after the DNA fragments had been subcloned into the M13 phage. The plasmids constructed were designated according to the oligonucleotides used as the reverse primers.

Table I. Oligonucleotide primers used for constructing deletions at the COOH-terminal end of the wild-type beta -subunit protein


Oligonucleotide Nucleotide sequencea Carboxy-terminal amino acidb

RQ1-f2 5'-GTTACACATTCGAGCTCGAGTCGC-3'
RQ1-DL512 5'-CGAGATCTAGAGGTCGTAGAGATACG-3' Leu-512
RQ1-DS534 5'-CGAGATCTAAGAATCGCAACCCGATG-3' Ser-534
RQ1-DS555 5'-CGAGATCTAAGACCTGCTTATCTTCG-3' Ser-555
RQ1-DK558 5'-CGAGATCTATTTGCCGGTAGACCTG-3' Lys-558
RQ1-DD560 5'-CGAGATCTAATCGAATTTGCCGGTAG-3' Asp-560
RQ1-DQ562 5'-CGAGATCTACTGTATATCGAATTTGCCGG-3' Gln-562
RQ1-DY563 5'-CGAGATCTAATACTGTATATCGAATTTG-3' Tyr-563
RQ1-DI564 5'-CGAGATCTAGATATACTGTATATC-3' Ile-564
RQ1-DA564c 5'-CGAGATCTACGCGTACTGTATATCGAATTTGC-3' Ala
RQ1-DL564c 5'-CGAGATCTAGAGATACTGTATATCGAATTTGC-3' Leu
RQ1-DA565 5'-CGAGATCTACGCGATATACTGTATATC-3' Ala-565
RQ1-DC566 5'-CGAGATCTAGCACGCGATATACTG-3' Cys-566
RQ1-DS567 5'-CGAGATCTAACTGCACGCGATATACTG-3' Ser-567
RQ1-DS568 5'-CGAGATCTAGCTACTGCACGCGATATAC-3' Ser-568
RQ1-DR569 5'-CGAGATCTAACGGCTACTGCACGCG-3' Arg-569
RQ1-DV570 5'-CGAGATCTAAACACGGCTACTGCAC-3' Val-570
RQ1-DA572 5'-CGAGATCTATGCCAGAACACGGCTAC-3' Ala-572
RQ1-DY574 5'-CGAGATCTAGTAGGGTGCCAGAAC-3' Tyr-574
RQ1-DV576 5'-CGAGATCTAGACCCCGTAGGGTGC-3' Val-576
RQ1-DQ578 5'-CGAGATCTATTGGAAGACCCCGTAG-3' Gln-578
RQ1-DL585 5'-CGAGATCTATAGAGACGCAACCTTC-3' Leu-585

a Underlined sequences indicate the termination signals in the reverse primers.
b Numbers indicate the amino acid positions in the wild-type beta -subunit protein, where the protein synthesis was terminated artificially at the mutant gene.
c An Ala or Leu residue was substituted for the Ile-564 residue of the DI564 protein.

In Vivo Complementation Analysis

E. coli 594/F' lacIq cells carrying the plasmids were grown at 37 °C in YT broth (0.8% tryptone (Difco), 0.5% yeast extract, 0.5% sodium chloride) containing 10 mM CaCl2 to a cell density of 3 × 108 cells/ml, at which time phage Qbeta sus51, which is defective in the beta -subunit gene, was added at a multiplicity of infection of 0.1. After 10 min at 37 °C, the cells were spun down and resuspended in an equal volume of YT broth. The cell suspension was then diluted 104-fold with YT broth containing 2 mM isopropyl-1-thio-beta -D-galactopyranoside, and the incubation was continued at 37 °C for 90 min, at which time chloroform was added. An aliquot (100 µl), after appropriate dilution, was immediately mixed with a large excess of a stationary culture of E. coli A/lambda or BE110 and then plated onto a YT broth-agar plate.

Preparation of Cell Lysates

E. coli 594/F' lacIq cells carrying the plasmids were grown in 5 ml of YT broth containing 50 µg/ml ampicillin to a cell density of 2 × 108 cells/ml, at which time 2 mM (final concentration) of isopropyl-1-thio-beta -D-galactopyranoside was added to the medium, and the incubation was continued for another hour. Cell lysates were prepared according to Ball and Kaesberg (17). Aliquots (5 µl) of the lysates were analyzed by SDS-polyacrylamide gel electrophoresis to demonstrate that a similar amount of beta -subunit protein was present in each lysate (Fig. 2). For the gel retardation assay, the lysates were centrifuged at 30,000 × g for 30 min at 4 °C, and the supernatants were used.


Fig. 2. Synthesis of the beta -subunit proteins in the cells carrying the beta -subunit gene with deletions. Lysates were prepared from E. coli 594/F' lacIq cells carrying pUC8 (lane b), pRQ1-DS534 (lane c), pRQ1-DS555 (lane d), pRQ1-DK558 (lane e), pRQ1-DD560 (lane f), pRQ1-DQ562 (lane g), pRQ1-DY563 (lane h), pRQ1-DI564 (lane i), pRQ1-DA565 (lane j), pRQ1-DC566 (lane k), pRQ1-DS567 (lane l), pRQ1-DS568 (lane m), pRQ1-DR569 (lane n), pRQ1-DV570 (lane o), pRQ1-DA572 (lane p), pRQ1-DV576 (lane q), pRQ1-DQ578 (lane r), pRQ1-DL585 (lane s), or pRQ1 (lane t). A 5-µl sample of each cell lysate was applied to a 7% SDS-polyacrylamide gel. Dots indicate the position of the beta -subunit protein. The Roman numerals next to lane a indicate the positions of the marker proteins: phosphorylase B (92.5 kDa) (I); bovine serum albumin (66.2 kDa) (II); ovalbumin (45.0 kDa) (III).
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Assay for Polymerase Activities Using Cell Lysates

The wild-type and mutant polymerase activities were measured using poly(rC), MDV-poly(+) RNA, or Qbeta RNA as described previously (10), with slight modifications. The standard reaction mixtures (50 µl) each contained 125 mM Tris-HCl (pH 8.0), 25 mM 2-mercaptoethanol, 10 mM MgCl2, 50 mM phosphoenol pyruvate, 10 µg/ml pyruvate kinase, 74 units/ml DNase I, 10 µg/ml rifampicin, and 5 µl of cell lysate. For the poly(rG) experiment, the mixture also included 0.4 mM GTP, 20 µg/ml poly(rC), and 740 kBq/ml [alpha -32P]GTP. The MDV-poly RNA or Qbeta RNA reaction mixture contained 0.8 mM each ATP, CTP, and GTP, 0.1 mM UTP, 20 µg/ml MDV-poly(+) RNA or 68 µg/ml Qbeta RNA, and 1.48 MBq/ml [alpha -32P]UTP. Incubation was performed at 35 °C for 15 min (MDV-poly RNA polymerase assay) or at 37 °C for 60 min (Qbeta RNA polymerase assay). Aliquots (10 µl) of the mixtures were applied to 3MM Whatman filter paper discs. Incorporated radioactivity was determined by liquid scintillation counting (10). In control reactions, 100 ng of purified Qbeta replicase was exogeneously added to the lysates of host cells carrying pUC8. From the control assay results, it was determined that 5 µl of the lysates of cells carrying pRQ1 contained the equivalent of 271 ng (Qbeta RNA polymerase activity) or 247 ng (MDV-poly RNA polymerase activity) of pure Qbeta replicase.

In this paper, MDV-poly(+) RNA represents an RNA that was transcribed from pUC-MDV-LR DNA by T7 RNA polymerase in vitro.

Northern Blot Analysis

E. coli 594/F' lacIq cells were grown and infected as described under "In Vivo Complementation Analysis" except that the phage was added at a multiplicity of infection of 3. After 10 min at 37 °C, the cells were spun down and resuspended in an equal volume of YT broth containing 2 mM isopropyl-1-thio-beta -D-galactopyranoside, and then the incubation was continued for the indicated times. RNAs were extracted with phenol and precipitated with ethanol in the presence of 0.8 M LiCl. The pellets were washed twice with 70% ethanol and then dissolved in distilled water. A 5-µg sample of the extracted RNA was denatured with glyoxal and dimethyl sulfoxide and then applied to a 1.0% agarose gel.

Electrophoresis, transfer to a nylon membrane (Biodyne A, Pall Ultrafine Corp.), and hybridization were performed as described previously (13).

A 1.2-kilobase pair XhoI-BamHI fragment of ZpQbeta -32 DNA, which lies within the maturation (A2) gene, was labeled using E. coli DNA polymerase I (Klenow fragment), random oligonucleotide primers (nonamers), and [alpha -32P]dCTP and then was used as the hybridization probe to detect Qbeta viral RNA.

Gel Retardation Assay

The 5'-end of MDV-poly(+) RNA was labeled as described previously (10). Ice-cold binding mixture (15 µl) comprised 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.1 mM dithioerythritol, 5% glycerol, 1.5 × 10-2 pmol of 32P-end-labeled MDV-poly(+) RNA, and 1 µl of various cell lysate supernatants. After 20 min of incubation at 5 °C, samples (10 µl) were applied to a 5% polyacrylamide gel at 4 °C as described by Werner (18). The gel was then dried and subjected to autoradiography.

Purification of the Replicase

The wild-type and DS567 replicases were purified from E. coli 594/F' lacIq cells carrying the plasmids according to Kajitani and Ishihama (19). For the Qbeta RNA polymerase assay, the standard reaction mixtures (50 µl) each contained 80 mM Tris-HCl (pH 7.5), 12 mM MgCl2, 12 mM 2-mercaptoethanol, 0.8 mM each ATP, CTP, and GTP, 0.1 mM UTP, 74 kBq of [alpha -32P]UTP, and 200 ng of the replicase. Incubation was performed at 37 °C for 15 min. Incorporated radioactivity was determined as described under "Assay for Polymerase Activities Using Cell Lysates."


RESULTS

Phage Growth in Cells Carrying the Plasmids

We first investigated whether or not the mutated beta -subunit proteins could complement the activity of phage Qbeta sus51 replicase, an amber mutant of the beta -subunit gene. E. coli 594/F' lacIq cells carrying the mutant plasmids were infected with phage Qbeta sus51, and then proliferation of the phage in the cells was examined.

As shown in Fig. 3A, as long as the amino acid deletion was less than 19 residues from the carboxyl-terminal end of the beta -subunit protein, cells carrying each plasmid, pRQ1-DL585, -DQ578, -DV576, -DA572, and -DV570, produced as many progeny phages as did cells carrying the wild-type plasmid pRQ1 (Fig. 3A). However, when the deletion extended beyond the Val-570 residue, progeny production steadily decreased. Furthermore, when the deletion reached the Ile-564 residue, the phage production in cells carrying larger deletion plasmids was abolished completely.


Fig. 3. Effects of amino acid deletions on Qbeta replicase activity. A, in vivo complementation activity of the cells carrying the mutant plasmids. The percentage of activity was calculated on the basis of the phage titer for cells carrying pUC8. The frequency of phage adsorption was more than 80% for every clone. B, in vitro RNA polymerase activity. The relative RNA polymerase activity (ng/reaction) of cell lysates was determined on the basis of the amount of [32P]UMP incorporated in the reaction mixture containing 100 ng of purified Qbeta replicase using Qbeta RNA (darkened bars) or MDV-poly(+) RNA (open bars) as a template. Values are the means ± S.D. for two independent determinations; bars indicate S.D. Footnote 1, lysate of cells carrying pUC8. Footnote 2, lysate of cells carrying pRQ1.
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Northern Blot Analysis

To investigate the cause of the poor phage production in cells carrying mutant plasmids (Fig. 3A), we extracted RNAs from phage Qbeta sus51-infected cells, and subjected the RNAs to Northern blot analysis.

As shown in Fig. 4, the 32P-labeled Qbeta cDNA probe strongly hybridized with Qbeta phage RNA (Fig. 4, lane a) but did not hybridize with cellular RNAs at all (Fig. 4, lane b), demonstrating that this cDNA probe specifically detected Qbeta viral RNA. In the control cells carrying pRQ1, viral RNA synthesis occurred at 20 min after the Qbeta sus51 infection, and many viral RNAs accumulated during another 20 min of incubation (Fig. 4, lanes i and j). On the other hand, in the cells carrying plasmid pRQ1-DA565 or -DS567, in which phage production was greatly reduced (Fig. 3A), viral RNA was found only a little even at 40 min after the infection (Fig. 4, lanes e-h). Since viral RNA synthesis did not occur in the cells carrying pUC8 (Fig. 4, lanes c and d), the residual viral RNA syntheses found in the cells carrying the mutant plasmids were not due to the invading phage genomes.


Fig. 4. Northern blot analysis of RNAs from cells infected with phage Qbeta sus51. Phage Qbeta sus51 was introduced into E. coli 594/F' lacIq cells carrying pUC8 (lanes c and d), pRQ1-DA565 (e and f), pRQ1-DS567 (g and h), or pRQ1 (i and j) and incubated for another 20 min (c, e, g, and i) or 40 min (d, f, h, and j). Lane b represents mock infection of cells carrying pUC8. Five-µg samples of RNAs were applied to the gel, except for in lane a, where 12 µg of Qbeta phage RNA was applied. 16 and 23 S rRNAs of E. coli MRE600 were electrophoresed in a lane on the gel, transferred to a nylon membrane, and their positions were determined by cutting the lane of the membrane and staining it with methylene blue.
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Also seen were the hybridization signals at the positions near to those of 16 and 23 S rRNAs (Fig. 4, lanes f and h-j). These signals may represent the premature transcripts of Qbeta RNA because such small RNAs were observed in Qbeta RNA-dependent RNA synthesis by wild-type or DS567 replicase (see Fig. 5C).


Fig. 5. Effects of the ratio of Qbeta RNA to replicase on the Qbeta RNA-dependent RNA synthesis. A, time course of RNA synthesis. The reaction mixture (50 µl) contained 0 µg (shaded symbols), 0.016 µg (open symbols), or 1 µg (closed symbols) of Qbeta RNA and 500 ng of wild-type (circles) or DS567 (squares) replicase. Values are the means ± S.D. for two independent determinations; bars indicate S.D. B, relationships between the molar ratio (Qbeta RNA/replicase) and Qbeta RNA synthesis. The reaction mixture (50 µl) contained 200 ng of wild-type (circles) or DS567 (squares) replicase and the indicated amounts of Qbeta RNA. Incubation was performed at 37 °C for 10 min. 200 ng of the replicase was equivalent to 0.66 pmol of the holoenzyme, and 1 µg of Qbeta RNA was equivalent to 0.70 pmol of the RNA. C, gel electrophoresis of the RNAs synthesized. The RNA synthesis was carried out at 37 °C for 10 min (lanes a and c) or 20 min (lanes b and d). Equal amounts of radioactive RNA (1,100 cpm) synthesized by DS567 (lanes a and b) or wild-type (lanes c and d) replicase at the molar ratio of 5 were denatured and applied to each lane of a 1% agarose gel. After electrophoresis, RNAs were transferred to a membrane (Biodyne A). The positions of Qbeta RNA, 16 and 23 S ribosomal RNAs were determined as in Fig. 4.
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Qbeta RNA Polymerase Activity

Using lysates from cells carrying various mutant plasmids described above, we examined Qbeta RNA-dependent RNA polymerizing activity.

As shown in Fig. 3B, as long as the amino acid deletion was less than 18 residues, the polymerizing activity of lysates from the cells carrying the mutant plasmid was similar to that of the control cell lysate. However, when the deletion extended beyond the Val-570 residue of the beta -subunit, despite the fact that the cells expressing the mutant replicases supported poor growth of the invading Qbeta sus51 phage (Fig. 3A), lysates of the cells carrying pRQ1-DR569, -DS568, -DS567, or -DC566 synthesized much more RNA than did lysate from cells harboring pRQ1. These results suggest that those deletions did not affect the polymerizing activity of the replicase. When 25 or more amino acids were deleted from the carboxyl terminus of the beta -subunit protein, polymerizing activity was abolished. In addition, lysates of cells carrying pUC8 exhibited little RNA polymerizing activity.

To further analyze the characterization of the mutant replicase, we purified DS567 replicase and compared the Qbeta RNA-dependent RNA synthesis by wild-type and DS567 replicases. As shown in Fig. 5A, when 16 ng of the template Qbeta RNA was added to the reaction mixture, DS567 replicase synthesized RNA in a similar manner and amount to wild-type replicase. However, when 1 µg of the template RNA was incubated with DS567 replicase, incorporation of [32P]UTPs was about 2-fold of that using wild-type replicase. Fig. 5B shows the relationships between Qbeta RNA-dependent synthesis and the molar ratio of Qbeta RNA to replicase (Qbeta RNA/replicase). Since Qbeta replicase binds to Qbeta RNA at three sites (5), an excess amount of Qbeta RNA causes binding of Qbeta replicase to two or three Qbeta RNA molecules, resulting in inhibition of Qbeta RNA synthesis (20). Under our experimental conditions, Qbeta RNA-dependent synthesis was found to be maximum at a molar ratio of about 0.3 or 0.5 for the wild-type or DS567 replicase reaction, respectively. When the ratio was higher than 0.5, DS567 replicase synthesized RNA twice as much as wild-type replicase did, which was consistent with the results in Fig. 4A. When the 32P-labeled RNAs synthesized by wild-type or DS567 replicase at a molar ratio of 0.5 were compared by an agarose gel electrophoresis, most of the 32P-labeled RNAs were found to be similar in size to Qbeta viral RNA (Fig. 5C). These results indicate that the polymerization activity of DS567 replicase did not get damaged and suggest that DS567 replicase had an extra ability to bind to Qbeta RNA.

Template Specificity of the Mutant Replicases

It was puzzling that lysates of cells carrying pRQ1-DC566, -DS567, -DS568, and -DR569 exhibited higher polymerizing activity in vitro with the Qbeta RNA template but that these cells were unable to support effectively the proliferation of the invading phage, Qbeta sus51.

Taking account of the results in Fig. 5, the mutant replicases are assumed to have more relaxed template specificity than the wild-type replicase, so thereby they neglect Qbeta RNA synthesis in cells infected with phage Qbeta sus51. Therefore, we examined the polymerizing activities of cell lysates using cellular RNAs as templates. As shown in Table II, lysates of cells carrying pRQ1-DC566, -DS567, -DS568, or -DR569 incorporated more [32P]UTPs than the control cell lysates carrying pRQ1 when ribosomal RNA or total cellular RNA was used as template. In contrast, lysates of cells carrying pRQ1-DA572 or -DV576, which exhibited lower Qbeta RNA polymerase activity in vitro but supported progeny production in Qbeta sus51-infected cells, had lower polymerization activity than the control lysate in the cellular RNA-dependent system.

Table II. Cellular RNA-directed RNA synthesis by cell lysates from cells carrying the mutant plasmids


Template RNA [32P]UMP incorporated by cell lysatesa
Vectorb DA565 DC566 DS567 DS568

pmol
Qbeta RNAc 0.2  ± <0.1 18.4  ± 4.5 50.2  ± 5.6 70.6  ± 5.4 46.1  ± 1.6
rRNAsd 0.1  ± <0.1 0.2  ± <0.1 0.8  ± <0.1 3.0  ± 0.1 1.8  ± <0.1
total RNAse 0.1  ± 0.1 0.3  ± <0.1 1.0  ± 0.2 3.3  ± 0.6 2.3  ± 0.6
DR569 DV570 DA572 DV576 Wild typef

Qbeta RNAc 27.2  ± 0.9 18.1  ± 0.1 10.5  ± 0.2 8.2  ± 0.3 16.2  ± 2.2
rRNAsd 0.8  ± 0.2 0.7  ± 0.1 0.4  ± <0.1 0.1  ± <0.1 0.6  ± 0.2
total RNAse 0.9  ± 0.1 0.9  ± 0.2 0.4  ± 0.1 0.2  ± 0.1 1.1  ± 0.6

a Values are the means ± S.D. for two independent determinations.
b Lysate from cells carrying pUC8.
c Data from Fig. 3B.
d 16 and 23 S ribosomal RNAs (2 µg) of E. coli MRE600 were used in each reaction.
e Total cellular RNAs were extracted from E. coli 594/F' lacIq cells carrying pUC8, and 4 µg of the RNAs were used in each reaction.
f Lysate from cells carrying pRQ1.

To further determine the cause of the difference between the in vivo and in vitro replicase reactions, we compared the template specificity of purified wild-type and DS567 replicases. Qbeta replicase transcribes closely related phage RNAs but not other RNAs such as ribosomal RNAs and unrelated phage RNAs (5). As shown in Table III, when SP RNA, a closely related phage RNA to Qbeta RNA, was also used as a template, DS567 replicase incorporated more [32P]UTPs than the wild-type replicase, as in the case of Qbeta RNA template. DS567 replicase also exhibited much higher RNA polymerizing activity for 16 and 23 S ribosomal RNAs or total cellular RNA as template, confirming the results of Table II. These results indicate that DS567 replicase relaxed specificity for not only Qbeta RNA template but also cellular RNA templates.

Table III. Comparison of the template specificity between wild-type and DS567 replicases


Replicase [32P]UMP incorporated with a templatea
Qbeta RNA SP RNA 16 and 23 S rRNAs Total cellular RNAsb  -RNA

pmol
Wild type 94.5  ± 4.2 60.9  ± 10.6 1.2  ± 0.2 2.0  ± <0.1 <0.1
DS567 176.2  ± 20.3 99.9  ± 9.9 2.5  ± <0.1 4.1  ± 0.2 0.1 ± 0.1

a Values are the means ± S.D. for two independent determinations. The reaction mixture (50 µl) contained 1 µg of Qbeta RNA or SP RNA, 2 µg of 16 and 23 S rRNAs of E. coli MRE600, or 4 µg of total cellular RNAs as a template.
b RNAs were extracted from E. coli 594/F' lacIq cells carrying pUC8.

MDV-poly RNA and Poly(rG) Polymerase Activities

Since Qbeta replicase lacking S1 and HF-I does not transcribe Qbeta RNA but can use MDV-1 RNA and poly(rC) as template, we examined the effects of deletions on MDV-poly RNA and poly(rG) polymerase activities. The MDV-poly(+) RNA-dependent polymerizing activity of cell lysates changed in a similar way to Qbeta RNA-dependent polymerizing activity as the amino acid deletion extended into the inner part of the beta -subunit protein (Fig. 3B). When 25 residues were deleted, lysates of cells expressing mutant replicases with larger deletions no longer showed the polymerizing activity.

The poly(rC)-dependent polymerizing activity of cell lysates carrying pRQ1-DI564 decreased by 75% compared with the control cell lysates, as in the case of the MDV-poly(+) RNA template (Table IV). Furthermore, replacement of the carboxyl-terminal Ile residue of DI564 replicase with Ala or a chemically similar Leu residue reduced the poly(rG) and MDV-poly(+) RNA polymerizing activities to background levels. These results indicate that the sequence from Ile-564 to Ala-565 of the beta -subunit protein is important for both the MDV-poly RNA and poly(rG) polymerase activities.

Table IV. Effects of amino acid substitutions on MDV-poly RNA and poly(rG) polymerase activities


Cell lysate [32P]UMP or [32P]GMP incorporated with a templatea
MDV-poly(+) RNA poly(rC)

pmol
Vectorb 0.3  ± 0.2 0.6  ± 0.8
DY563 0.6  ± 0.1 1.0  ± 0.1
DI564 11.3  ± 1.6 25.6  ± 3.0
DA564 0.8  ± 0.4 2.0  ± 1.3
DL564 2.6  ± 0.6 4.4  ± 0.4
DA565 59.0  ± 5.1 78.8  ± 3.5
Wild typec 83.0  ± 15.1 96.5  ± 9.5

a Values are the means ± S.D. for two independent determinations.
b Lysates of cells carrying pUC8.
c Lysates of cells carrying pRQ1.

MDV-poly(+) RNA Binding Activity

Qbeta replicase binds to the central region of MDV-1(+) RNA, in which nucleotides 81-126 are identical with nucleotides 84-129 of Qbeta minus-strand RNA (21). To determine whether or not mutant replicases without MDV-poly RNA polymerizing activity can bind to a template, we examined a gel retardation assay using 32P-end-labeled MDV-poly(+) RNA. When the lysates of cells carrying pRQ1-DA565, pRQ1-DC566, or pRQ1 were incubated with [32P]MDV-poly(+) RNA and then electrophoresed on a polyacrylamide gel, the mobility of [32P]MDV-poly(+) RNA was decreased (Fig. 6, lanes c and g-i). In contrast, in the case of lysates of cells carrying pRQ1-DI564, which exhibited poor polymerizing activity (Fig. 3B and Table IV), only a small amount of [32P]MDV-poly(+) RNA migrated slowly (Fig. 6, lane f), indicating that the binding of MDV-poly(+) RNA to DI564 replicase was greatly impaired. Furthermore, in the case of pRQ1-DQ562 or -DY563, a mobility shift was not observed anymore (Fig. 6, lanes d and e).


Fig. 6. Binding of various replicases to MDV-poly(+) RNA. 32P-End-labeled MDV-poly(+) RNA (1,000 cpm) was mixed with the lysis buffer (lane a) or the lysate supernatants of cells carrying pUC8 (lane b), pRQ1 (lanes c, i, and j), pRQ1-DQ562 (lane d), pRQ1-DY563 (lane e), pRQ1-DI564 (lane f), pRQ1-DA565 (lane g), or pRQ1-DC566 (lane h). In lane j, 2 pmol of nonlabeled MDV-poly(+) RNA was added to the reaction solution. The arrow indicates the position of MDV-poly(+) RNA.
[View Larger Version of this Image (94K GIF file)]


DISCUSSION

Qbeta replicase distinguishes its own Qbeta RNA and closely related viral RNAs among natural RNAs. This discrimination involves the recognition of a specific secondary or tertiary structure of the template RNA. Qbeta RNA provides the replicase with three binding sites (S-site, the initiation region of the coat gene; M-site, the internal region of the beta -subunit gene; and the 3'-terminal region of Qbeta RNA) (3). However, little is known about the counterparts of the replicase molecule responsible for the enzyme specificity, such as template recognition or subunit assembly. In this paper, we propose that the carboxyl-terminal part of the beta -subunit of Qbeta replicase is one of the candidates for functional domains determining the template recognition from the following results.

Qbeta RNA Polymerizing Activity

DS567 replicase with deletions of 21 amino acid residues at the carboxyl terminus had a relaxed template specificity for a related phage RNA and cellular RNAs as well as Qbeta RNA (Tables II and III, Fig. 5). Furthermore, lysates of cells expressing DR569, DS568, DS567, or DC566 replicase, which showed in vitro higher polymerizing activity with the Qbeta RNA template but failed to support progeny production in the cell, exhibited higher polymerizing activity in cellular RNA-dependent synthesis (Table II). In contrast, lysates of cells expressing DA572 or DV576 replicase showed lower polymerizing activity than the control lysate in Qbeta RNA or cellular RNA-dependent synthesis (Table II); however, these mutant proteins complemented the activity of phage Qbeta sus51 replicase (Fig. 3A). These results suggest that in cells infected with phage Qbeta sus51, a mutant replicase having a more relaxed template specificity than that of wild-type replicase engaged in transcribing cellular RNAs as well as Qbeta RNA and accordingly failed to perform the task of polymerizing Qbeta RNA, indicating that the accessibility of Qbeta replicase to cellular RNAs may be critical for phage growth.

MDV-poly(+) RNA Polymerase Activity

DI564 replicase lacking the carboxyl-terminal 24 amino acid residues of the beta -subunit protein showed only a little activity in the MDV-poly RNA polymerase assay (Fig. 3B). On removal of the terminal Ile residue from DI564 replicase, the resulting mutant, DY563, replicase lost the polymerizing activity (Table IV). Furthermore, DI564 replicase showed reduced binding ability to MDV-poly(+) RNA, and DY563 replicase abolished it completely (Fig. 6). These results indicate that the failure of MDV-poly(+) RNA-dependent RNA synthesis by DI564 replicase was due to poor binding of the replicase to the RNA. Therefore, the sequence from Ile-564 to Ala-565 of the beta -subunit protein was essential for Qbeta replicase to express MDV-poly RNA polymerase activity, particularly for its binding to MDV-poly(+) RNA. The finding that the insertion of a pentapeptide at the Ala-565 residue abolished the MDV-1 RNA polymerizing activity of Qbeta replicase (6) agrees with our results.

Recently, Brown and Gold (22) have proposed a three-site model of Qbeta replicase, according to which two RNA binding sites, site I on the S1 subunit and site II on EF-Tu, are responsible for RNA binding, and the polymerase-active site on the beta -subunit is for RNA synthesis. Site I binds class I RNAs that possess single-stranded regions containing a high fraction of A and C nucleotides such as Qbeta plus-strand RNA, and site II binds class II RNAs that have polypyrimidine tracts such as Qbeta minus-strand RNA or MDV-1 RNA. According to their model, destruction of site II or removal of EF-Tu from Qbeta replicase will result in the replicase without the MDV-1 RNA binding activity. Therefore, the data in Fig. 6 that show the importance of the Ile-564 and Ala-565 residues of the beta -subunit protein in MDV-poly(+) RNA binding suggest that site II on EF-Tu interacts with these amino acid residues to form an active Qbeta replicase molecule.

Our present results indicate that a change in the carboxyl-terminal structure of the beta -subunit protein of Qbeta replicase could cause relaxation of the template specificity and that this part of the beta -subunit is responsible for recognizing MDV-poly(+) RNA. The carboxyl-terminal region of the beta -subunit protein is heterologous in RNA coliphages. Phage RNA replicase is thus assumed to have evolved its template specificity in part by altering the carboxyl-terminal structure of the beta -subunit and simultaneously by keeping the accessibility of the replicase to cellular RNAs at a low level.


FOOTNOTES

*   This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence and reprint requests should be addressed. Tel.: 81-28-627-7213; Fax: 81-28-627-7187.

ACKNOWLEDGEMENTS

We thank Akikazu Hirashima for reading the manuscript and for helpful discussions. We also thank our colleagues in our laboratory for help in isolating the mutant replicase clones.


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