tRNATrp as Primer for RNA-directed DNA Polymerase: Structural Determinants of Function*

The specific interactions between the RNA-directed DNA polymerase of avian oncornavirus and the tRNATTp primer required for initiation of viral DNA synthesis in vitro were examined. Two distinct interactions, stable binding of the tRNATrp to the enzyme and initiation of viral DNA synthesis by the enzyme with tRNA’s’P as primer, were characterized as to the structure of tRNA’r’P required. Different structural features of the tRNATP were shown to be necessary for each type of interaction. The entire primary structure and native conformation of tRNAlb are both required for binding to reverse transcriptase. Fragments of tRNA’r’P and intact tRNAlkp in an altered conformation cannot be bound by the enzyme using an assay which detects high affinity binding between reverse tran- scriptase and native tRNATrp. In contrast, fragments of the tRNA’rrP molecule can serve as primers for viral DNA synthesis with normal efficiency as compared to intact tRNA’r’P. The fragments which initiate transcrip- tion must contain a minimum specific nucleotide sequence which extends from the 3’ terminus of the tRNATrp through 27 residues of the molecule. This por- tion of the tRNA’m may be a major structural determinant

The specific interactions between the RNA-directed DNA polymerase of avian oncornavirus and the tRNATTp primer required for initiation of viral DNA synthesis in vitro were examined.
Two distinct interactions, stable binding of the tRNATrp to the enzyme and initiation of viral DNA synthesis by the enzyme with tRNA's'P as primer, were characterized as to the structure of tRNA'r'P required. Different structural features of the tRNATP were shown to be necessary for each type of interaction.
The entire primary structure and native conformation of tRNAlb are both required for binding to reverse transcriptase. Fragments of tRNA'r'P and intact tRNAlkp in an altered conformation cannot be bound by the enzyme using an assay which detects high affinity binding between reverse transcriptase and native tRNATrp. In contrast, fragments of the tRNA'rrP molecule can serve as primers for viral DNA synthesis with normal efficiency as compared to intact tRNA'r'P. The fragments which initiate transcription must contain a minimum specific nucleotide sequence which extends from the 3' terminus of the tRNATrp through 27 residues of the molecule. This portion of the tRNA'm may be a major structural determinant of specificity in initiation.
Transcription of DNA in vitro from the native genome of avian oncornaviruses by the viral RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) initiates on a molecule of tRNA""' that is bound to a specific site near the 5' terminus of the viral genome (l-4). Although a number of other isoacceptor species of tRNA are also associated with the genomes of these viruses (5), tRNA"""' is responsible for most if not all of the initiations by the viral polymerase ("reverse transcriptase") (6). Removal of tRNA""" from the viral genome abolishes template activity for the polymerase (7). Panet et al. (8) demonstrated stable and highly specific binding between the reverse transcriptase of avian myeloblastosis virus and tRNA""" and proposed that this binding mediates the specific initiation of transcription from the viral genome. In an effort to test this hypothesis, we prepared fragments of tRNA""' and compared the abilities of these fragments to bind to reverse transcriptase and to serve as primers for transcription from the genome of avian sarcoma virus. We also tested the interaction between polymerase and two other forms of RNA: (i) a duplex of 16 pairs of complementary nucleotides adjoining the 3' terminus of the tRNA molecule that binds tRNA"'p to the genome of ASV' (9,10) and (ii) tRNA""" whose conformation was altered by basepairing with a complementary nucleotide sequence from the ASV genome. Our results indicate that the stable binding of tRNA""' to RNA-directed DNA polymerase of ASV requires both the native configuration and virtually all of the primary structure of the tRNA molecule. By contrast, only a portion of the molecule is required for initiation of DNA synthesis; the nucleotide sequence of this portion may be a major determinant of specificity in initiation.

EXPERIMENTAL PROCEDURES
Materials-The sources of most materials have been described previously (5,11 Gigh molecular weight subunits were then separated fro& ihe low molecular weight RNAs by gel filtration through agarose (A-15m) as described previously (15). Purification of Primer from ASV-tRNA""' was purified by twodimensional electrophoresis in slab gels (12 X 17 cm); the first dimension was in 10% polyacrylamide for 3.0 h at 350 V, the second in 20% polyacrylamide for 16 h at 350 V. This procedure has been described in detail (5,16). tRNA"""' labeled with ,"P was located in the slab gel by autoradiography, excised, and eluted by shaking the crushed gel in ' The abbreviation used is: ASV, avian sarcoma virus. Isolation of the RNase-resistant Duplex between tRNA"" and the ASV Genome-["'P]tRNA"'" was reannealed with high molecular weight ASV subunit RNA as previously described (15 Nucleotide Sequence Analysis-"'P-labeled primer and primer fragments were hydrolyzed with RNase TI (Calbiochem), and the resulting oligonucleotides were separated by two-dimensional paper electrophoresis as described by Sanger et al. (19). Individual oligonucleotides from fingerprints were redigested with RNase A and the products were analyzed by electrophoresis on DEAE-paper at pH 3.5 (20,21 (22). The remainder of each fraction was assayed for the presence of ""P-labeled RNA by measurement of Cerenkov radiation in a liquid scintillation counter. To recover the radioactive RNA from the Sephadex column buffer, pooled fractions were diluted 2-fold with water and passed over a 0.2ml DEAE-cellulose column equilibrated in water.
The RNA was eluted with 0.5 ml of 2 M NaCl. The sample was dilute-d to 1 ml with water and precipitated with 2 volumes of ethanol.

RESULTS
Enzymatic Fragmentation of tRNATrJ'-Partial hydrolysis of tRNA'rrP with RNase TI produced at least 10 distinct fragments that could be separated by electrophoresis in a polyacrylamide gel ( Fig. la; two fragments obtained in relatively low yield B and D are not visible in the reproduction). Each of the fragments was composed of a unique nucleotide sequence, as determined by two-dimensional "fingerprinting" following exhaustive hydrolysis with RNase TI. We also determined the composition of each oligonucleotide in the fingerprints; these data allowed us to locate the fragments within the established nucleotide sequence of tRNA'rrP (Figs. 1  b, 3'-and 5'-terminal halves of primer from cleavage by S1 nuclease. '"P-labeled tRNA""' was cleaved by S1 nuclease as described under "Experimental Procedures." The cleavage products were separated by electrophoresis in a 20% polyacrylamide slab gel and isolated as described above. Nucleotide sequence analysis was used to identify each fragment; the results are portrayed schematically. by guest on May 12, 2020 http://www.jbc.org/ Downloaded from 2). As a set, the fragments characterized represented all portions of the tRNA except a sequence of 19 nucleotides located at the 5' end of the molecule (Figs. 1 and 2).
Under suitable conditions, S1 nuclease cleaves tRNA molecules only in the anticodon and in the nucleotide sequence C-C-Aou at the 3' terminus (17,18). We cleaved tRNA""" in this manner, separated the resulting molecular halves by electrophoresis in a polyacrylamide gel (Fig. lb), and then identified the halves by analysis of nucleotide sequence (Fig.   2).
Fragments of tRNATrp Do Not Bind to Reverse Transcriptase-Panet et al. (8) detected binding of tRNA'n" to reverse transcriptase by filtration of reaction mixtures through columns of Sephadex G-100. We employed the same procedure in our studies and, in preliminary experiments, duplicated the previous findings. When filtered separately through the column, tRNA and polymerase eluted at different positions; polymerase activity was detectable only in the void volume of the column, well ahead of the retarded tRNA. By contrast, when polymerase and tRNA'rrP were mixed prior to filtration, they eluted together in the void volume of the column; in these and all of the following experiments, we used polymerase in IOO-fold molar excess of RNA in order to obtain maximum binding of RNA to enzyme.
In accord with previous results (B), we found that the reverse transcriptase of ASV specifically bound tRNA"""'; with the exception of tRNAdM", other isoacceptor species of tRNA found in virions of ASV did not bind (data not illustrated).
By contrast, specificity of this high order was not observed with another assay for binding, i.e. a filter binding assay (25).' Using a filter binding assay, reverse transcriptase readily binds to a variety of viral and nonviral RNAs. Therefore, we employed a stringent assay in the present studies in order to distinguish the exceptional affinity of reverse transcriptase for tRNA""' from less stable interactions between the polymerase and RNA.
In an effort to identify specific portions of the tRNA'l"'" molecule that interact with reverse transcriptase, we subjected the tRNA to partial hydrolysis by RNase T, and then tested the resulting fragments of RNA for their ability to bind to the polymerase. The hydrolyzed RNA (Fig. 4, Lane I) contained both residual intact tRNA""' and a set of fragments similar to that illustrated in Fig. 1. Approximately half of the nucleasetreated tRNA bound to reverse transcriptase and eluted in the void volume of the Sephadex column (Fig. 3A). Analysis by electrophoresis in a polyacrylamide gel revealed that the bound RNA was composed of intact tRNA (Fig. 4, Lane 2) and tRNA""" molecules lacking adenosine from the 3/-C-C-ASH terminus (data not shown). However, no fragments were detected even after prolonged autoradiography of the gel. In order to test fragments for binding in the absence of appreciable amounts of intact tRNA, we recovered the unbound RNA from the column and subjected it to a second cycle of binding. Only a small fraction of the RNA bound to reverse transcriptase (Fig. 3B), and this RNA proved to be a trace amount of intact tRNA that had failed to bind in the initial reaction (Fig. 4, Lane 3; RNA not visible in the reproduction). The unbound RNA was composed entirely of fragments (Fig. 4, Lane 4) conclusion, we examined the interaction between polymerase and a mixture of 3' and 5' halves of tRNA""'" prepared by hydrolysis with S, nuclease as described above (Fig. lb). The RNA was denatured to assure separation of the molecular halves and then mixed with reverse transcriptase under conditions for binding. Approximately 30% of the RNA bound to polymerase (data not illustrated), and this RNA consisted only of unfragmented tRNA molecules (Fig. 4, Lane 5); the unbound RNA was composed of the 3' and 5' halves of the tRNA (Fig. 4, Lane 6). We conclude that a single interruption in the primary structure of tRNA""' (except for the removal of the 3'-terminal C-C-Aou (17,18)) can ablate the ability of this RNA to bind to reverse transcriptase.
The Duplex Formed Between a Portion of tRNATr/' and a Nucleotide Sequence in the Genome of ASV Does Not Bind to Reverse Transcriptase-A sequence of at least 16 base pairs binds tRNA""' to the genome of ASV (9,lO). This duplex begins with the penultimate nucleotide at the 3' terminus of the tRNA and is therefore immediately adjacent to the site where DNA synthesis initiates; consequently, we hypothesized that the duplex might bind reverse transcriptase as a prerequisite for initiation of DNA synthesis. We prepared the duplex as described previously (9 scribed previously (9). The purified duplex failed to bind to reverse transcriptase (data not shown).
Not all Conformations of tRNATrp Can Bind to Reverse Transcriptase-We have found that the tertiary conformation of tRNA'rrP is altered when the RNA is bound to the ASV genome by hydrogen-bonded base pairs; annealing of tRNA'rrP to the viral genome exposes new sites on the tRNA molecule to cleavage by S1 nuclease." Since the tRNA functions as primer while base-paired with the viral genome, we tested the ability of tRNA'rrP in the base-paired form to bind to reverse transcriptase. A substrate suitable for this test was prepared as follows. The duplex, composed of the base-paired region between primer and viral genome, was isolated from the unlabeled native genome of ASV, using hydrolysis with RNase and filtration through agarose as described above. The unlabeled duplex was then denatured and reassociated in the presence of ["'P]tRNA'rrP, transcriptase both before and after denaturation of the RNA ( Fig. 6A and B, respectively).
Although the reassociated form of primer was poorly resolved from polymerase activity in the assay (Fig. 6A), binding of RNA to enzyme was not apparent. By contrast, release of primer from the reassociated form by denaturation resulted in extensive binding (Fig. 6B); primer released from the reassociated form by denaturation had the electrophoretic mob& ity of native tRNA'rrP (Fig. 5, Lane 2). These findings suggest that the interaction between reverse transcriptase and native tRNArrp is not identical with the interaction between the polymerase and the primer on the genome of ASV. In order to test this issue further, we performed the experiments described in the following section.

Fragments of tRNATrJ' as Primers for DNA Synthesis-
The data presented above indicate that the binding of tRNA'rrP to reverse transcriptase requires the native structure of the tRNA. In order to determine whether similar requirements govern the initiation of DNA synthesis, we tested fragments of tRNATrp as primers for the transcription of DNA from the ASV genome.
Nine of the fragments generated by partial hydrolysis of tRNA'rrP with RNase T1 contain the 3' terminus of the tRNA and part or alI of the adjacent nucleotide sequence that basepairs with the viral genome, Fragments A, B, C, D, E, F, and J ( Fig. la). In order to confirm the identity of these fragments, we exploited our previous observation that the 3' terminus of tRNA'rW can be "tagged" with radioactive dAMP, the nucleotide that initiates transcription from the native ASV genome (11). "Tagged" primer was cleaved with RNase T1 and the radiolabeled fragments were separated by electrophoresis in a polyacrylamide gel (Fig. 7, Lane I). Corresponding The duplex formed between tRNArv and the genome of ASV was prepared (9) using unlabeled nucleic acids. The purified duplex was denatured by boiling in water for 5 min, then annealed with [:'2P]tRNA"q' as described under "Experimental Procedures." Annealing was carried out with the unlabeled duplex in approximately 20-fold molar excess over the labeled tRNA'rT'. minal fragments were generated as a subset of the total fragments by hydrolysis of uniformly labeled tRNA"'" (Fig. 7, Lane 2). One of the 3'-terminal fragments (I) co-migrates with a major internal fragment (Z) and was, therefore, detected only by the device of "tagging" the 3-terminal fragments with radioactive dAMP.
We evaluated the ability of 3'-terminal fragments to form stable duplexes with the genome of ASV by annealing a mixture of uniformly labeled fragments with denatured viral RNA. Fragments bound to the viral genome after the annealing were isolated by fiitration through a column of agarose 15m (15), released from viral RNA by denaturation, and then analyzed by electrophoresis in a polyacrylamide gel (Fig. 7, Lane 4). Only the shortest 3'-terminal fragment (L) failed to bind to the viral genome. Small amounts of the slightly longer fragment, denoted K, were detectable in the bound RNA but are not easily visible in the reproduction, and the remainder of the 3'-terminal fragments all bound to viral RNA in readily detectable amounts.
We then tested whether any of the 3'-terminal fragments could serve as primers for transcription of DNA from the genome of ASV. We cleaved unlabeled tRNA"'" with RNase T1, annealed the resulting fragments of RNA with the denatured genome of ASV, and used the annealed RNA as template-primer for reverse transcriptase with E""P]dATP as the only precursor. Fragments A through F were "tagged" by this procedure (Fig. 7, Lane 3) and, therefore, had served as primers for the polymerase. By contrast, Fragments G through K had no detectable primer activity, although all of these fragments possess the 3' terminus of intact tRNAl"p and can form stable base pairs with viral RNA.
In order to further substantiate the significance of these findings, we tested the primer activity of fragments in reactions with polymerase as a limiting reagent. These tests were designed on the basis of data generated by varying the amount of polymerase added to reaction mixtures containing a constant amount of template-primer.
The primer activity of fragments was tested as described above, using one saturating concentration and two different limiting concentrations of polymerase, 35% and 55% saturation; in all three instances, L-FIG. 7. Identification of fragments from tRNA"'" that can initiate DNA synthesis by the DNA polymerase of ASV. Samples of '"Plabeled RNAs were analyzed by electrophoresis in a slab gel of 20% polyacrylamide for 4 h at 400 V and 15°C. Lan.e I, identification of fragments that include the 3' terminus of tRNA""'. the same set of fragments initiated DNA synthesis with for initiation of DNA synthesis. In order to measure the roughly comparable efficiencies as judged from the relative efficiency of primer activity, we purified Fragments A and C intensities of the bands on the autoradiogram (Fig. 8). of tRNArrp and then measured the ability of each of these The preceding experiments provided only a qualitative test fragments to base-pair with the genome at ASV and to initiate transcription of DNA (Table I). Both of the fragments initi-1 2 3 ated DNA synthesis with efficiencies approximating the efficiency obtained with complete tRNATrp. We attempted to measure the primer efficiency of several smaller fragments as well, but we were unable to obtain these in sufficient purity to permit decisive measurements. \ DISCUSSION We have identified structural features of tRNA'nP that are required for two distinct interactions with the reverse transcriptase of ASV, stable binding of the tRNA to the enzyme, and initiation of DNA synthesis by the enzyme with the tRNA j as primer. Our data indicate that both the entire primary ,' * structure and the native tertiary configuration of the tRNA 1 I .' .,i are probably required for binding to polymerase, whereas a '_ ,\ ! ." ,* much smaller but specific fraction of the tRNA molecule can Neither form of RNA bound to reverse transcriptase. We presume that the duplex structures tested by us failed to bind because they either did not duplicate all of the essential features of the native template-primer or because the binding constants of these structures are lower than the assay can detect.
The affinity between certain tRNAs and the reverse transcriptase of ASV could account for at least two features of virion structure. First, both tRNA'n" and tRNAdML.' are relatively abundant within virions of ASV (29). Since both tRNAs bind to reverse transcriptase with exceptional affinity (8), and since virions contain roughly equivalent numbers of molecules of polymerase (30) and of the two tRNAs (29), it is possible that binding to reverse transcriptase may facilitate incorporation of these tRNA's into the vii-ion. Second, formation of base pairs between tRNA'r'P and the genome of ASV requires disruption of an appreciable portion of the secondary structure of the native tRNA. Perhaps the binding to reverse transcriptase denatures the tRNA and, therefore, facilitates formation of base pairs with the viral genome.
Our data define a sequence of nucleotides within tRNA"'" that is sufficient to serve as primer for transcription of DNA from the genome of ASV. The requirement for this sequence appears to be stringent, elimination of two nucleotides from its 5' end reduces or eliminates primer function (compare Fragments F and G, Fig. 7, Lane 3), although appreciably shorter sequences (e.g. Fragments H, I and J) form stable duplexes with the ASV genome and provide the proper 3' terminus for initiation of DNA synthesis. Residues 59 through 75 (see Fig. 9) of the required sequence are nucleotides known to be involved in base-pairing with the ASV genome (9, 10). All but one of the remaining nucleotides in the required sequence (residues 50 to 58, Fig. 9) may also base-pair with viral RNA (28); although this base-pairing has yet to be demonstrated experimentally, it could conceivably account for the role of this portion of the tRNA molecule in the initiation of DNA synthesis.
The primer on the genome of the Moloney strain of murine leukemia virus is tRNA"" (31), which can bind to and initiate DNA synthesis by the reverse transcriptase of ASV (26). The only substantial homology between the nucleotide sequence of tRNA"" and that of tRNAT'" is found in residues 48 to 58, C-G-U-G-$-G-C-G-m'A-Ap for tRNA'r'", C-G-G-G-++C-Am'A-Ap for tRNA'"'.4 This similarity corroborates our conclusion that these nucleotides are required for the initiation of DNA synthesis.
Both tRNA"'" and tRNA"" have 2 adjacent residues of pseudouridine in loop IV of the cloverleaf replacing the characteristic doublet of ribothymidine and pseudouridine common to most tRNAs (3,31,32). Consequently, it has been proposed that these nucleotides are involved in the interaction between the tRNAs and reverse transcriptase (26). Our data neither refute nor confirm this possibility, but we can conclude that the presence of the pseudouridines does not suffice for initiation of DNA synthesis by the polymerase; fragment H contains both the 3' terminus of tRNA"'" and the 2 residues of pseudouridine, yet fails to serve as primer for reverse transcriptase (Fig. 7, Lane 3).
Panet et al. (8) have proposed that the stable binding of reverse transcriptase to tRNA'l"" mediates the initiation of transcription from the genome of avian oncornaviruses. The data presented here cast some doubt on this proposal since fractions of tRNA7"" that fail to bind to reverse transcriptase can nevertheless serve as primers for site-specific initiation of DNA synthesis by the polymerase.
We conclude that stable binding between reverse transcriptase and primer is not necessarily a prerequisite for sitespecific initiation of transcription from retrovirus genomes. Only a portion of the tRNA""' is required for initiation of viral DNA synthesis by the polymerase, and the nucleotide sequence of this portion may be a major structural determinant in directing the specificity in initiation.