Reverse transcriptase mediated binding of primer tRNA to the viral genome

Abstract

A complex between tRNATrp (beef) and 35 S RNA from avian myeloblastosis virus is obtained when the mixture is preincubated in the presence of reverse transcriptase at 35 degrees C. The tRNA-RNA complex is active in initiating DNA synthesis catalyzed by reverse transcriptase. The interaction of tRNA with reverse transcriptase involves the partial unwinding of the acceptor stem of tRNA, as evidenced by nuclease digestion with RNAase T1 and micrococcal nuclease. When tRNA2Glu (coli), having a high degree of similarity with primer tRNA at the level of the acceptor stem, was used as primer for DNA synthesis, a low but significant level of incorporation was obtained, if the reaction was performed at 35 degrees C, while a high incorporation, similar to the one obtained with tRNATrp was obtained when the annealing between tRNA2Glu and 35 S RNA was performed at 80 degrees C. Our evidences point out to an important role of the viral DNA polymerase in positioning the primer on the RNA genome.

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

“…Previous reports demonstrated that AMV reverse transcriptase forms a stable and specific complex with the replication primer tRNATrp (Panet et al, 1975;Araya and Litvak, 1979) and is able to partially unwind the acceptor stem of the tRNA favouring annealing of primer tRNA Tp to the RSV genome (Araya et al, 1979). Here we provide biochemical and genetic data indicating that finger protein RSV p12 or MuLV plO directs the annealing of the replication tRNA primer onto the primer binding site of the viral genome under physiological conditions.…”

“…The function of the small finger proteins can be understood if both the nucleic acid unwinding property and the cis-acting sequences of the target RNAs are taken into account. RSV leader (or MuLV leader) and replication primer tRNA are both highly structured at 37°C (Panet et al, 1975;Araya and Litvak, 1979;Fournier et al, 1976;Darlix et al, 1986) and hybridization seems unlikely without destruction of the secondary structures. Unwinding of these RNA structures, which promotes the base pairing interactions between complementary sequences (e.g.…”

Structural features required for the binding of tRNATrpto avian myeloblastosis virus reverse transcriptase

“…It has been proposed that this interaction is responsible for presence of a specific subset of host tRNAs in ALSV particles (18)(19)(20)(21). Reverse transcriptase binding may be required for hybridization of the primer tRNA to the genomic template RNA (22).…”

The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNALys3 to HIV-1 genomic RNA

“…One possibility, suggested by in vitro studies, is that RT plays a direct role in tRNA Lys3 annealing. Early work indicated that the in vitro annealing of primer tRNA Trp to AMV genomic RNA was promoted by the addition of AMV reverse transcriptase [23]. In a later work, in which it was demonstrated that HIV-1 RT interacted with the D arm and TΨC loop of tRNA Lys3 , HIV-1 RT was also shown facilitate the in vitro annealing of tRNA Lys3 to the PBS sequence [24].…”