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Volume 270, Number 2, Issue of January 13, 1995 pp. 581-588
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Formation of the 2`,5`-Phosphodiester Linkage in the cDNA Priming Reaction by Bacterial Reverse Transcriptase in a Cell-free System (*)

(Received for publication, September 16, 1994; and in revised form, November 2, 1994)

Tadashi Shimamoto (§), ,  Masayori Inouye,  Sumiko Inouye (¶)

From the Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2`,5`-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2`,5`-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2`,5`-phosphodiester linkage in msDNA synthesis is carried out by RT itself.

INTRODUCTION

Reverse transcriptases (RT) (^1)are unique among DNA polymerases because of their ability to use RNA as templates. Eukaryotic RTs associating with retroviruses and retrotransposons are known to use a specific cellular tRNA as a primer for cDNA synthesis(1) . Recently it has been demonstrated that the 3`-OH group of the 3`-end A residue of tRNA is used exclusively for the priming reaction by HIV retroviral RT(2) . In contrast to these eukaryotic RTs, it has been suggested that bacterial RTs specifically initiate cDNA synthesis from the 2`-OH group of an internal G residue of a template RNA(3, 4) .

Bacterial RTs have been shown to exist in a Gram-negative soil bacterium, Myxococcus xanthus(5, 6) , and Escherichia coli(7, 8) . Bacterial RTs homologous to retroviral RTs are responsible for the synthesis of an unusual satellite single-stranded DNA called msDNA (multicopy single-stranded DNA). The 5`-end of msDNA is covalently linked to the 2`-OH group of an internal G residue of a single-stranded RNA (msdRNA). The DNA and RNA molecules form a heteroduplex at their 3`-ends. A number of different msDNAs have been found in Myxobacteria, E. coli, Rhizobium, Salmonella, Proteus, Klebsiella(3, 4, 9, 10) . A genetic element called ``retron'' is required for msDNA production and consists of msr (a coding region for msdRNA), msd (a coding region for msDNA), and RT (a gene for RT)(3) . Eight different retrons have been so far identified and characterized in E. coli and Myxobacteria.

The proposed synthesis of msDNA is shown in Fig. 1(3, 4) . First, an RNA transcript encompassing msr, msd, and RT is produced from the promoter located upstream of msr (step 1 in Fig. 1). The RNA transcript containing a1 and a2 inverted repeat sequences is thought to form secondary structures including a stable a1-a2 stem structure (step 2). The branching G residue is placed at the end of the a1-a2 stem in this folded structure. No specific primary sequences are required in the a1-a2 structure(11, 12) . In order for RT to initiate msDNA synthesis, another specific structure for each RT located downstream of the branching G residue is required in addition to the a1-a2 stem structure and the G residue(12) . The primary reaction of msDNA or cDNA synthesis is thought to start from the 2`-OH group of the G residue (step 3). The first base is added using the RNA transcript as the template, and cDNA synthesis continues on the same template RNA to a specific termination site (steps 3 and 4). The template RNA is removed by RNase H, leaving the short 3`-end RNA-DNA overlapping region.

Figure 1: Biosynthetic pathway of msDNA synthesis. Short thin arrows represent the inverted repeats (a1-a2 and b1-b2). Thick arrows represent the genes for msdRNA (msr), msDNA (msd), and RT. The branching G residue is circled. Long solid lines represent mRNA transcript. Thick lines in the transcript correspond to msdRNA, and wavy lines correspond to cDNA or msDNA. See text for details.


In this report, we purified a bacterial RT to homogeneity from E. coli harboring the RT gene from retron-Ec73. Using the purified enzyme (RT-Ec73) and an RNA template synthesized in vitro by T7 RNA polymerase (Fig. 2A), we established a cell-free system synthesizing the full-length msDNA-Ec73 (Fig. 2B). Furthermore, we now unambiguously demonstrate that the bacterial RT is indeed able to prime cDNA synthesis from the 2`-OH group of a specific internal G residue of the template RNA molecule. Therefore, the cDNA-priming mechanism of bacterial RTs appears to be quite different from that of retroviral RTs. This raises interesting questions as to the molecular mechanism and the evolutionary significance of the 2`-OH priming reaction.

Figure 2: Proposed secondary structures of msr-msd RNA from retron-Ec73 synthesized in vitro by T7 RNA polymerase and msDNA-Ec73. A, putative secondary structure of msr-msd RNA from retron-Ec73 synthesized in vitro by T7 RNA polymerase using pUCT7MS73 as a template. The branching G residue is circled. The two arrows indicate a1-a2 inverted repeats. A solid triangle indicates a termination site of msDNA-Ec73 synthesis in vivo and an open triangle indicates a cleavage site of the template RNA by RNase H after msDNA synthesis (see Fig. 2B). The synthesized RNA is 192 bases in length. B, secondary structure of msDNA-Ec73(19) . The branching G residue is circled, and the RNA region is boxed. The trinucleotide (5`-AGC-3`) covered with a shaded box is linked to cDNA after digestion with RNase A.


EXPERIMENTAL PROCEDURES

Bacterial Strains

In order to purifiy RT-Ec73(His) protein, the T7 expression system was used(13) . A K-12 derivative containing DE3 phage, which contains the T7 RNA polymerase gene under the control of the lac promoter, was constructed. Using E. coli strain LE392 (supE44, supF58, hsdR514, galK2, galT22, metB1, TrpR55, lacY1) a DE3 lysogen was isolated as described previously(14) . The new -lysogen was designated LE392(DE3) and used as a host for the T7 expression system. All phage manipulations were done according to Sambrook et al.(15) . CL83 (16) was used as a host to manipulate plasmids.

Construction of Plasmids

pET73RT(His), which carries the RT-Ec73 gene under the control of a T7 promoter for high expression of the protein (13) and a sequence encoding a histidine-tag (17) at the N terminus of RT-Ec73, was constructed as follows. First, pET11a(Km) was constructed by inserting the 1.3-kilobase HincII-EcoRI fragment of the kanamycin-resistant gene from Tn5 (18) into the DraI-EcoRI sites of pET11a(13) . pUC7XbaI was constructed by inserting a oligonucleotide, 5`-GATCCTCTAGACCCGGGTCTAGAG-3`, into the BamHI site of pUC7. Then, 1,059-base pair XbaI fragment including the RT-Ec73 gene from the mutation 5 (19) of retron-Ec73 was inserted into the XbaI site of pUC7XbaI, and a resulting plasmid was designated pUC7Xba-RT. The 824-base pair NdeI-BamHI fragment containing the downstream part of the RT-Ec73 gene was cut out from pUC7Xba-RT and inserted into the NdeI and BamHI sites of pET11a(Km). The resulting plasmid was designated pET73RTDeltaN. The upstream part of the RT-Ec73 gene containing the histidine-tagging sequence and an NdeI site was made by polymerase chain reaction using oligonucleotides 73nh (5`-TCCATATGCATCACCATCACCATCACAGAATATATAGCCTA-3`) and 73n-1 (5`-AATGCATATGCAGCA-3`) as primers, and inserted into the SmaI site of pUC19. The sequence of the inserted fragment was confirmed by the dideoxy sequencing method(20) . The NdeI fragment was then cut out and ligated into the NdeI site of pET73RTDeltaN. pET73RT(His) was isolated by screening a plasmid which had the insert in the correct orientation. To check whether pET73RT(His) produced the active RT-Ec73(His), LE392(DE3) was cotransformed with pET73RT(His) and p73-Hc0.7 which carries only the msr-msd region from retron-Ec73(12) . After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the transformant, msDNA was isolated as a plasmid fraction according to the alkaline-sodium dodecyl sulfate (SDS) method (21) and analyzed by polyacrylamide gel electrophoresis. The production of msDNA-Ec73 was confirmed (data not shown).

pUCT7MS73 was constructed for in vitro transcription. First, an 84-base pair fragment including a T7 promoter (Ø10) was amplified by polymerase chain reaction using oligonucleotides T7p-73 (5`-CTAGGTTTGGCTCTGCTATAGTGAGTCGTA-3`) and PBR-b (5`-CCGGCCACGATGCGTCC-3`) as primers and pET11a (13) as a template. A G residue was used as the first base for transcription in front of the 5`-end C residue of msr because the T7 RNA polymerase preferably uses G for the transcription initiation(22) . After purification by polyacrylamide gel electrophoresis, this fragment was mixed with p73-Hc0.7, and the second polymerase chain reaction was done by using the PBR-b and 73f (5`-TCGGATCCTTATGCACCTT-3`) (12) as primers. The amplified fragment was isolated and cloned into the SmaI site of pUC19(23) . A plasmid, which had the insert in the opposite orientation against the lac promoter of pUC19, was selected and designated pUCT7MS73. The DNA sequence of the inserted fragment was confirmed by the method of Sanger et al.(20) .

Purification of RT-Ec73(His)

LE392(DE3)/pET73RT(His) cells were grown in M9 medium supplemented with 0.2% Casamino Acids (Difco), 0.4% glucose, 20 µg/ml tryptophan, 2 µg/ml thiamine, 0.8 mM MgSO(4), and 50 µg/ml kanamycin at 37 °C up to 90 Klett units. Then IPTG was added to a final concentration of 1 mM. After 1 h of induction, the cells were harvested and washed once with 50 mM Tris-HCl (pH 7.5) (lane 2 in Fig. 3). Then the cells were resuspended in French press buffer (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM beta-mercaptoethanol, 1 mM MgCl(2), and 10% glycerol) and disrupted by passing through a French press cell. Unbroken cells were precipitated by low speed centrifugation (4,000 times g, 10 min). The membrane and soluble fractions were then separated by ultracentrifugation (135,000 times g, 30 min) (lane 4). The overexpressed RT-Ec73(His) protein was fractionated into the membrane fraction. Thus, the membrane fraction was washed twice with French press buffer. RT-Ec73(His) was then solubilized from 20 mg of protein of membrane fraction in 2 ml of solubilization buffer (100 mM sodium-phosphate (pH 8.0), 10 mM Tris-HCl (pH 8.0), 6 M guanidine-HCl, and 2 mM beta-mercaptoethanol). The solubilized fraction was obtained by ultracentrifugation (135,000 times g, 30 min) (lane 5) and loaded to 1 ml of Ni-nitrilotriacetic acid (NTA) affinity resin (Qiagen). All column manipulations were performed under the denaturing condition at 4 °C by using the fast protein liquid chromatography system (Pharmacia). First the column was washed with 10 ml of elution buffer (7 M urea, 100 mM sodium-phosphate (pH 8.0), 10 mM Tris-HCl (pH 8.0), and 2 mM beta-mercaptoethanol) and 20 ml of elution buffer containing 20 mM imidazole. RT-Ec73(His) was eluted with 10 ml of elution buffer containing 100 mM imidazole. The eluted fractions (1 ml each) were dialyzed independently to refold the denatured protein as follows. Six peak fractions were first dialyzed against dialysis buffer A (50 mM Tris-HCl (pH 7.5), 5 mM beta-mercaptoethanol, 10% glycerol, 0.1% Nonidet P-40, and 0.5 M ammonium sulfate) containing 2.5 M urea at 4 °C for 3 h, and then dialysis buffer A containing 1 M urea for 3 h, the same buffer containing 0.5 M urea for 3 h and without urea for 3 h. Finally, all fractions were dialyzed against dialysis buffer B (50 mM Tris-HCl (pH 7.5), 5 mM beta-mercaptoethanol, 10% glycerol, 0.1% Nonidet P-40, and 200 mM NaCl) for 3 h. Protein aggregates were then precipitated by centrifugation (13,500 times g, 15 min). Protein patterns were analyzed by SDS-polyacrylamide gel electrophoresis (24, see Fig. 3), and RT activity was checked in a cell-free system as described below. Three peak supernatant fractions (1 ml each) were pooled (lane 6) and used for all experiments as the purified RT-Ec73(His).

Figure 3: Purification of RT-Ec73(His). RT-Ec73(His) was purified as described under ``Experimental Procedures.'' Lane 1, total cell protein of LE392(DE3)/pET73RT(His) without IPTG induction; lane 2, with IPTG induction; lane 3, the soluble protein fraction after high speed centrifugation; lane 4, the membrane protein fraction; lane 5, protein fraction solubilized with guanidine-HCl; lane 6, purified RT-Ec73(His) from Ni-NTA column. An arrowhead indicates the band corresponding to RT-Ec73(His). The calculated molecular mass of RT-Ec73(His) is 37.6 kDa. Bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa) were used as molecular mass markers. The gel was stained with Coomassie brilliant blue.


Preparation of the msr-msd RNA by T7 RNA Polymerase

First, pUCT7MS73 was digested with BamHI to linearize, then extracted with phenol and chloroform, precipitated with ethanol, and redissolved in 1 mM EDTA (pH 8.0). 10 µg of the linearized pUCT7MS73 was mixed with transcription buffer (40 mM Tris-HCl (pH 7.5), 6 mM MgCl(2), 2 mM spermidine, and 10 mM NaCl), 10 mM DTT, 0.5 mM each of NTP (ATP, GTP, CTP, and UTP) and 200 units of T7 RNA polymerase (Promega) to a total volume of 500 µl. The reaction mixture was incubated at 37 °C for 2 h and then extracted with phenol and chloroform. A half volume of 7.5 M ammonium acetate and three volumes of ethanol were added, and the mixture was placed at -70 °C for 30 min and then centrifuged (13,500 times g, 10 min). By this step, unincorporated nucleotides were removed. The precipitated RNA fraction was redissolved in 1 ml of 1 M ammonium acetate and divided to 10 portions. Three volumes of ethanol were added, and tubes were stored at -70 °C separately until needed. Immediately before use for cell-free synthesis of msDNA, the RNA was precipitated by centrifugation. The transcription buffer and DTT solution were supplied by Promega.

Cell-free Synthesis of msDNA-Ec73 in Vitro

The msr-msd RNA synthesized by T7 RNA polymerase in vitro (corresponding to 50 µl of reaction) was redissolved in 60 µl of annealing buffer (50 mM Tris-HCl (pH 8.0) and 10 mM MgCl(2)). 3 µl of the RNA solution was used for each reaction. To cause the RNA to fold properly, each sample was boiled for 2 min, incubated at 37 °C for 30 min and at 4 °C for 30 min before the reaction. The RNA thus treated was mixed with RT buffer (50 mM Tris-HCl (pH 7.8), 10 mM DTT, 10 mM MgCl(2), 60 mM NaCl, and 0.05% Nonidet P-40), 0.3 mM each of dGTP, dATP, dCTP, and 10 µCi of [alpha-P]dTTP (3,000 Ci/mmol). 3 µl (0.15 µg) of the purified RT-Ec73(His) was added to make the final volume of 25 µl, and the reaction mixture was incubated at 37 °C for 20 min. Then 1.5 µl of 5 mM dTTP was added to the mixture, and the reaction was continued at 37 °C for another 20 min. The reaction was stopped by adding 50 µl of stop solution (20 mM EDTA and 0.5% SDS) and 3 volumes of ethanol. The precipitated sample was redissolved in 10 µl of 1 mM EDTA and divided to two portions. One of them was heat-denatured and treated with RNase A (0.4 µg) for 10 min at 37 °C. The labeled products with and without RNase A treatment were separated by 6% polyacrylamide, 8 M urea gel.

To characterize the cDNA products, cDNA was synthesized in a large scale and labeled at the 3`-end. 120 µl of the RNA transcript was first annealed and mixed with RT buffer and 0.3 mM (each) dNTP. cDNA synthesis was started by adding 400 µl (20 µg) of the purified RT-Ec73(His) to 3 ml of reaction mixture and stopped by adding the stop solution and 3 volumes of ethanol. The precipitated sample was redissolved in 1 mM EDTA and loaded on polyacrylamide gel for electrophoresis. All bands were cut out of the gel and electroeluted to remove unincorporated nucleotides. The eluted products were labeled at the 3`-end in 200 µl of labeling buffer containing 250 µCi of [alpha-P]ddATP (5,000 Ci/mmol) and 96 units of terminal deoxynucleotidyl transferase (TdT, International Biotechnologies, Inc.) at 37 °C for 1 h as described previously (12) . The reaction was stopped with EDTA and precipitated with isopropyl alcohol. The labeled products were redissolved in 10 µl of 1 mM EDTA and heat-denatured. RNA was digested by incubating with 0.8 µg of RNase A at 37 °C for 10 min twice. Then the labeled products were separated on a preparative sequencing gel (12% polyacrylamide gel in 8 M urea). Each labeled band was cut out and eluted from the gel as described previously(12) . The DNA sequences were determined by the method of Maxam and Gilbert(25) .

Digestion of the cDNA Products with Debranching Enzyme and Ligation with Oligonucleotides

To carry out the debranching and ligation experiment, first band 4 and 5 products were repurified by urea/polyacrylamide gel electrophoresis and chromatography through a DE52 column (Whatman). The repurified products were dissolved in 20 µl (each) of the debranching reaction buffer (20 mM HEPES-HCl (pH 7.6), 40 mM KCl, 1 mM DTT, 0.5 mM MgCl(2), and 10% glycerol), and 0.17 µg of the purified yeast debranching enzyme (from Dr. J. D. Boeke) was added to the mixture and incubated at 30 °C for 1 h. 5 µl each of band 4 and 5 products with or without debranching enzyme treatment were annealed with oligonucleotides A (5`-CAAACCTAC-3`) and B (5`-CGTGCTCAAGTAGGTTTG-3`) in 10 µl of TM buffer (20 mM Tris-HCl (pH 7.6) and 10 mM MgCl(2)) as shown in Fig. 7A. The annealed oligonucleotides were mixed with ligation buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl(2), 10 mM DTT, and 0.5 mM ATP) and 1 unit of T4 DNA ligase and incubated at 14 °C overnight. All products were separated by sequencing gel (8 M urea, 20% polyacrylamide gel).

Figure 7: Analysis of the phosphodiester linkage between RNA and cDNA. A, schematic diagram of the digestion of the band 4 product with yeast debranching enzyme and ligation with the oligonucleotides. The branching G residues are circled, and RNA regions are boxed. Boldface letters represent cDNA product synthesized in the cell-free system. Asterisks indicate the P-labeled dideoxyadenosine. B, digestion of cDNA products with yeast debranching enzyme and ligation of resulted products with oligonucleotides. Experiments were carried out as described under ``Experimental Procedures.'' Band 4 product (lanes 1-4) and band 5 product (lanes 5-8) in Fig. 5were used in this analysis. The products digested with yeast debranching enzyme from band 4 and 5 are indicated by arrowheads in lanes 2 and 6, respectively. The digests (lanes 4 and 8) and undigested products (lanes 3 and 7) were ligated with oligonucleotides A and B. The ligated products from band 4 and 5 are indicated by arrowheads in lanes 4 and 8, respectively. Numbers in the left hand side indicate bases in length.


Figure 5: Analysis of cDNA products labeled at their 3`-ends. cDNAs were first synthesized in the cell-free system without any radioactive nucleotide and then labeled at their 3`-ends with [alpha-P]ddATP as described under ``Experimental Procedures.'' The synthesized cDNA products were divided to two parts, and each part was loaded to one lane of 12% polyacrylamide, 8 M urea preparative sequencing gel. Numbers with arrowheads represent the fragments used to determine DNA and RNA sequences (see Fig. 68). Molecular weight standards were the same as those described in Fig. 4.


Figure 6: DNA sequences of the cDNA products synthesized in the cell-free system. DNA sequences were determined by the method of Maxam and Gilbert(25) . Lane N, the same samples without sequencing reaction. A, DNA sequence of band 1 in Fig. 5. Dotted lines between the two gels indicate the same bases. B, DNA sequence of band 2 in Fig. 5. C, DNA sequence of band 3 in Fig. 5.


Figure 4: cDNA synthesis in vitro by using the purified RT-Ec73(His) and RNA template (msr-msd) synthesized in vitro. cDNAs were synthesized as described under ``Experimental Procedures'' were treated without (lane 1) or with (lane 2) RNase A and separated on 6% polyacrylamide, 8 M urea gel. The predicted structures of band a (lane 1) and bands b and c (lane 2) were depicted in the right hand side of each band (see ``Results'' for details). Products in band X are assumed to result from further cDNA extension of the band b product. Molecular weight standards were pBR322 digested with MspI and labeled at the 3`-ends with [alpha-P]dCTP and Klenow fragment of E. coli DNA polymerase I. Both thick and thin lines represent RNA template, where the thick region corresponds to msdRNA. Wavy lines represent cDNA. The branching G residue is circled.


RNA Sequence Analysis

To determine the sequence of the RNA portion of the cDNA products, band 4 product was treated with RNase T1 and repurified by urea/polyacrylamide gel. Note that the length of band 4 product after RNase T1 treatment was the same as before treatment. The repurified band 4 product was first labeled at the 5`-end of the RNA portion using T4 polynucleotide kinase (Boehringer Mannheim) and [-P]ATP (6,000 Ci/mmol), and the labeled product was repurified by urea/polyacrylamide gel. The labeled and repurified band 4 product was digested with the purified yeast debranching enzyme as described above. Band 4 products with or without debranching enzyme treatment were digested with RNase T1 or RNase U2 and separated by 8 M urea, 20% polyacrylamide gel electrophoresis as shown in Fig. 8. To compare the size of the RNA portion from band 4 product with that from msDNA-Ec73 produced in vivo, msDNA-Ec73 was isolated from CL83 harboring p23S3.5 (12) as described previously(12) , labeled at the 5`-end of the RNA, and digested with debranching enzyme followed by RNase T1 or RNase U2 in the same way as described above.

Figure 8: RNA sequence analysis by RNase digestion. Band 4 product in Fig. 5was first labeled with [-P]ATP and T4 polynucleotide kinase at the 5`-end of RNA portion. Then the labeled product digested with debranching enzyme (lanes 2, 4, and 6) or without digestion (lanes 1, 3, and 5) was treated with RNase T1 (lanes 3 and 4) or U2 (lanes 5 and 6). Each product was separated on 20% polyacrylamide, 8 M urea gel electrophoresis. Asterisks indicate P labeling. Note that the trinucleotide (5`-AGC-3`) migrated at exactly the same position as that from msDNA-Ec73 isolated in vivo (data not shown).


Other Materials

All restriction endonucleases were purchased from New England Biolabs, Life Technologies, Inc., or Boehringer Mannheim. T4 DNA ligase was obtained from Boehringer Mannheim. Taq DNA polymerase was from Perkin-Elmer Cetus. All radioactive products were purchased from Amersham Corp.

RESULTS

Construction of a Complete Cell-free Synthesis of msDNA-Ec73

In order to unambiguously demonstrate that bacterial RTs are able to prime cDNA synthesis from a specific internal G residue of a template RNA, we established a cell-free system for the production of msDNA. For this purpose, we first purified RT-Ec73 with a histidine tag (RT-Ec73(His)) to homogeneity from E. coli cells, LE392(DE3) harboring pET73RT(His), as described under ``Experimental Procedures'' (see Fig. 3). E. coli strain LE392(DE3), a K12 derivative, was created as a host strain to express RT-Ec73. Note that strain BL21(DE3) (14) commonly used as a host strain of T7 expression system is a derivative of E. coli B which has been known to contain retron-Ec86(8) . A template RNA was produced using T7 RNA polymerase in vitro as described under ``Experimental Procedures.'' The synthesized RNA is 192 bases in length and is thought to be folded as shown in Fig. 2A. Next, using the purified RT-Ec73(His) and the template RNA (msr-msd RNA), we examined the synthesis of msDNA, which was analyzed by urea/polyacrylamide gel as shown in Fig. 4. Without RNase A treatment, a broad band appeared at the position corresponding to single-stranded DNA markers between 270 and 220 in length (lane 1, band a). As shown later (see Fig. 5), band a products were found to consist of not only the fully extended msDNA linked to the intact RNA molecule but also short single-stranded DNA of approximately 10-15 bases in length also linked to the intact RNA molecule. Because of the absence of the RNase H activity in RT-Ec73(19) , all the cDNAs produced are considered to contain the full-length RNA template (see Fig. 4). After RNase A treatment, several bands appeared at position c (band c), which corresponds to a single-stranded DNA marker of about 16 bases. The bands above band c at about 20 bases are also considered to be products resulting from further extension of band c products. In addition, a higher molecular weight band (band b) was detected at a position corresponding to approximately 80 bases, which was almost identical to the position of the cDNA products synthesized from the RNA template isolated in vivo (data not shown). A higher molecular weight band (band X) also appeared at a position corresponding to about 90 bases. It is most likely that band X has longer cDNA extended beyond the termination site for band b (see Fig. 4). These data suggest that the present cell-free system is able to synthesize a full-length msDNA-Ec73.

DNA Sequence Analysis of the cDNA Products

To further characterize the cDNA products, a large scale production of msDNA was carried out as described under ``Experimental Procedures.'' The synthesized cDNAs were labeled at the 3`-ends with [alpha-P]ddATP and TdT. After RNase A treatment, they were separated by urea-polyacrylamide gel electrophoresis (see Fig. 5). The highest molecular weight band (band 1 in Fig. 5) migrated at a position a few bases larger than the expected product for msDNA-Ec73 produced in vivo, which migrates at the position of 76 bases. The RNase A treatment of msDNA-Ec73 isolated in vivo has been shown to result in a 73-base DNA attached to a 3-base RNA including the branching G residue(19) . The larger product was likely due to the extension by a few extra bases at the 3`-end. This was confirmed by DNA sequencing as described below. Most of the other products migrated at the positions shorter than a 27-base marker, indicating that most of the products resulted from premature termination. It appears that the cDNA synthesis stalled at the secondary structure corresponding to the msd stem region. However, once RT passes through this kinetic barrier, cDNA synthesis continued along the entire msd region.

Next, we determined DNA sequences of bands 1, 2, and 3 (Fig. 5) by the method of Maxam and Gilbert(25) . As shown in Fig. 6A, band 1 product had the same sequence as the in vivo msDNA-Ec73 (from T at position 1 to T at position 73, Fig. 2B) except for 2 or 3 extra A residues at the 3`-end. To determine whether the products migrating at positions shorter than the 27-base marker are produced by premature termination, the DNA sequences of band 2 and band 3 products (25 and 21 bases in length, respectively; Fig. 5) were determined. As shown in Fig. 6, B and C, the 5`-end of the DNA sequences of the band 2 and 3 products were 5`-TTGAGCACGTCGAT-3`, which is identical to that of msDNA-Ec73 produced in vivo (Fig. 2B) and the band 1 product (see Fig. 6A). The exact 3`-end sequences of the band 2 and 3 products were unable to be determined. These sequencing analyses clearly demonstrate that the cDNA synthesis very accurately started from the T residue complementary to the A residue at position 143 (Fig. 2A). Note that all samples without sequencing reaction (lane N in Fig. 6) migrated more slowly by one base than the other sequencing lanes. This is due to the piperidine treatment during sequencing reaction which eliminated one 5`-end base of the RNA molecule attached to DNA(12, 26) . Therefore the cDNAs synthesized in the cell-free system are likely to be linked to RNA.

Evidence for a 2`,5`-Phosphodiester Linkage between RNA and cDNA

In order to demonstrate that the cDNA products are linked to RNA by a 2`,5`-phosphodiester linkage as shown for the in vivo product, we used yeast debranching enzyme(27) , a specific nuclease to cleave a 2`,5`-phosphodiester linkage. Band 4 and 5 products migrating at 14 and 12 bases, respectively, were isolated from the gel in Fig. 5and digested with the purified yeast debranching enzyme as described under ``Experimental Procedures.'' Note that the sizes of the bands were estimated by the molecular weight markers used and Maxam-Gilbert sequencing ladders of band 1 product (data not shown). After treatment of band 4 and 5 products (Fig. 7B, lanes 1 and 5, respectively) with the debranching enzyme, the new bands appeared at 11 and 9 bases (lanes 2 and 6, respectively). The difference between before and after debranching enzyme reaction is 3 bases, which coincides well with the size of the RNA portion attached to msDNA-Ec73 (see Fig. 2B). It should be noted that the yeast debranching enzyme preferentially digests a substrate which has a purine residue at the 2`-end of the branch point; msDNA-Ec73 which has a T residue as the 2`-nucleotide is very poorly digested while msDNA-Ec86 which has a G residue as the 2`-nucleotide serves as a good substrate. (^2)

The 5`-end structures of both molecules were further examined by ligation analysis as depicted in Fig. 7A. Oligonucleotide B of 18 bases in length is designed to be complementary to both oligonucleotide A and the band 5 product in such a way that both oligonucleotides can be complementarily aligned on oligonucleotide B as shown in Fig. 7A. However, if the 5`-end of the band 5 product is blocked by a branched RNA molecule, the two oligonucleotides (oligonucleotide A and the band 5 product) cannot be ligated on oligonucleotide B. Indeed, the band 5 product, as well as the band 4 product, was unable to ligate to oligonucleotide A without treatment with debranching enzyme (Fig. 7B, lanes 7 and 3, respectively). On the other hand, when they were treated with the debranching enzyme, the 9- and 11-base molecules were generated (lanes 6 and 2, respectively) which were capable of ligating to the oligonucleotide A resulting in new longer products of 18 and 20 bases in length for bands 5 and 4, respectively (lanes 8 and 4). Debranching enzyme is known to generate a 5`-phosphoryl group(27) , which is consistent with the present result.

These data clearly demonstrate that both the band 4 and 5 products were blocked at their 5`-ends by forming a 2`,5`-phosphodiester linkage and had the identical sequence to the 5`-end sequence of msDNA-Ec73.

Determination of the RNA Sequence at the cDNA Priming Site

In order to determine whether the in vitro cDNA synthesis starts from the same G residue as that of msDNA-Ec73, we analyzed the RNA sequence attached to the 5`-end of the cDNA products. For this purpose, the band 4 product was treated with RNase T1 before and after the treatment of debranching enzyme. If the 2`-OH group of a G residue is blocked as a result of the DNA attachment, RNase T1 is not able to cleave at the G residue. The band 4 molecule labeled with [-P]ATP and T4 polynucleotide kinase at the 5`-end of RNA was purified by urea/polyacrylamide gel electrophoresis to remove the free radioactive nucleotide as described under ``Experimental Procedures.''

When the labeled band 4 product was digested with the yeast debranching enzyme, a new band (lane 2, Fig. 8) appeared at exactly the same position as the tri-ribonucleotide, 5`-AGC-3` that was derived by the same treatment from msDNA-Ec73 produced in vivo (data not shown). In order to determine the sequence of the tri-ribonucleotide from the band 4 product, it was further digested with RNase T1. As shown in lane 4, Fig. 8, a dinucleotide band was generated by RNase T1 treatment. It should be noted that the dinucleotide band was not obtained if the band 4 product was treated with RNase T1 before digestion with the debranching enzyme (lane 3). These results clearly indicate that the cDNA molecule was branched out from the 2`-OH group of a G residue. Furthermore, it was found that a mononucleotide was released by the treatment with RNase U2, a specific RNase for A residues, either before or after the debranching enzyme treatment (lanes 5 and 6). The mononucleotide released from the band 4 product was further confirmed as A by treating it with nuclease P1 followed by two-dimensional thin layer chromatography as described previously(26) . Together, the RNA sequence of the trinucleotide derived from the band 4 product was concluded to be 5`-AG(C or U)-3`, consistent with the sequence for msDNA-Ec73 (see Fig. 2B).

DISCUSSION

In this report, we constructed a complete cell-free system for the synthesis of msDNA-Ec73, which consists of the purified RT from retron-Ec73 (RT-Ec73), the msr-msd RNA template, and four dNTPs. Using this system, we now unambiguously demonstrate that the bacterial reverse transcriptase indeed initiates the cDNA priming reaction from the 2`-OH group of a specific internal G residue in the RNA template forming a 2`,5`-phosphodiester linkage at the 5`-end of the cDNA. The ability to form the 2`,5`-phosphodiester linkage is therefore an intrinsic property of the bacterial enzyme.

Bacterial RTs, although evolutionarily related to eukaryotic RTs, are significantly smaller than eukaryotic RTs. For example, RT-Ec73 consists of only 316 amino acid residues and has no RNase H domain (19) . Nevertheless, bacterial RTs have remarkably stringent requirements for the cDNA priming reaction. Each bacterial RT requires specific secondary structures downstream of the branching G residue for the cDNA priming reaction in addition to a stem structure immediately upstream of the G residue(11, 12) . Furthermore, we have recently demonstrated a requirement of secondary structures in the region serving as the cDNA template(28) . It is of great interest to determine how bacterial RTs are able to specifically recognize these RNA secondary structures. It has been shown that HIV-1 RT forms a heterodimer between the full-length 66-kDa product (p66) and the 51-kDa product (p51) lacking the C-terminal RNase H domain(29, 30) . The primer tRNA molecule is proposed to bind to the p51 subunit(31) . Recently, the requirements of secondary structures around the primer binding site have been reported for the cDNA priming reaction for retroviral RTs(32, 33, 34) . These requirements may somehow be related to those found for bacterial RTs in terms of the three-dimensional structures of the enzymes.

It is also an intriguing question how various RTs have developed their own specific cDNA priming mechanism; retroviral RTs use cellular tRNAs (1) , whereas RT from hepatitis virus (hepadnavirus) uses the protein itself as a primer for cDNA synthesis(35) . RT from a non-LTR retrotransposon, R2Bm, is known to have endonuclease activity, which creates a nick in double-stranded DNA to prime the reverse transcription of RNA template(36) . The Mauriceville plasmid RT initiates cDNA synthesis from the 3`-end of the template RNA without any specific primers as in the case of RNA-dependent RNA polymerase (37) .

The significance of the formation of the 2`,5`-phosphodiester linkage remains to be answered. Interestingly, the transposition efficiency of Ty1 element, a yeast retrotransposon, was significantly reduced in a strain lacking the debranching enzyme DBR1, suggesting that the formation of a 2`,5`-phosphodiester linkage may somehow be involved in the retrotransposition of Ty1 element(27) .

It is interesting to note that RNase H was not required for msDNA synthesis, although the addition of RNase H to the cell-free system stimulated the production of msDNA. (^3)The accumulation of msDNA of 10 15 bases in length observed in the present study may be due to the stable secondary structure in the RNA template, which hinders the elongation of cDNA synthesis. It is possible that another protein factor(s) such as an RNA helicase may be used for efficient production of msDNA in vivo.

FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM44012 and Takara Shuzo Co. Ltd. (Kyoto, Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Fellowship LT-225/92 from the International Human Frontier Science Program Organization.

To whom correspondence should be addressed: Dept. of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635. Tel.: 908-235-4161; Fax: 908-235-4783.

(^1)
The abbreviations used are: RT, reverse transcriptase; msDNA, multicopy single-stranded DNA; msdRNA, RNA molecule attached to msDNA; HIV, human immunodeficiency virus; IPTG, isopropyl-beta-D-thiogalactopyranoside; ddATP, dideoxyadenosine triphosphate; TdT, terminal deoxynucleotidyl transferase; DTT, dithiothreitol.

(^2)
K. Nam and J. D. Boeke, personal communication.

(^3)
T. Shimamoto, M. Inouye, and S. Inouye, unpublished data.

ACKNOWLEDGEMENTS

We thank Dr. Monica J. Roth for critical reading of this manuscript. We thank Dr. J. D. Boeke (Johns Hopkins University) for generous gift of the purified yeast debranching enzyme DBR1 and information about the enzyme before publication. We also thank Dr. F. W. Studier (Brookhaven National Laboratory) for the gift of all phage strains and M.-Y. Hsu for construction of pET11a(Km).

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