When retroviral reverse transcriptases reach the end of their RNA templates

Abstract

Luo and Taylor (J. Virol. 64:4321-4328, 1990) have previously shown that when, during RNA-directed DNA synthesis, a retroviral reverse transcriptase comes to a halt at the end of an RNA template, the associated RNase H produces a specific oligonucleotide that contains the 5' end of that template; in those studies the length of the oligonucleotide was predominantly 17 nucleotides. We have now investigated variables that might affect the formation and length of such a terminal oligonucleotide. We found small but significant variations in the length could be caused by the choice of reaction conditions and also the sources of reverse transcriptase and RNA template. Nevertheless, the general finding in all these situations was that RNase H acted at or about 14 to 18 nucleotides from the 5' end, thereby supporting the interpretation that in the reverse transcriptase, the cleavage site for the RNase H is held at around this distance behind the DNA polymerase activity. In other words, it appears that for the intact protein, the RNase H and reverse transcriptase activities may work in a coupled or coordinate manner. We also found that more than 80% of the residual 5' oligonucleotides remained base paired to the RNA-directed DNA product. Furthermore, under certain conditions, these short RNAs could act as efficient primers for an associated DNA-directed DNA synthesis in the reverse direction.

Escherichia coli RNase HI inhibits murine leukaemia virus reverse transcription in vitro and yeast retrotransposon Ty1 transposition in vivo

“…In vitro inhibition by E. coli RNase HI of cDNA synthesis by MuLV RT seems to occur via degradation of RNA-DNA hybrids that become accessible to E. coli RNase HI as a result of disassociation of RT from an incomplete cDNA-RNA hybrid. It was previously shown that RNA of RNA-DNA hybrids generated as reverse transcripts terminated with ddNTPs is degraded by the RNase H of RT 7-18 nucleotides (Oyama et al 1989;Schatz et al 1990;Fu & Taylor 1992) from the 3 0 -OH (sometimes 3 0 -H) of the DNA. The RNA remains associated with DNA since the RNA can be further degraded by E. coli RNase HI to even smaller products (Oyama et al 1989).…”

“…1). Two types of product might be left when RT leaves the first primer-template: an RNA-DNA hybrid whose length is that of the RNA copied into DNA, or a smaller RNA-DNA hybrid (7-18 bp) (Oyama et al 1989;Fu & Taylor 1992) resulting from cleavage of the RNA template by the RNase H of RT. Either of these products may serve as primer-template for a second RT.…”

“…RNase H activity of RT differs from that of E. coli RNase HI: (i) the specific activity of E. coli RNase HI is several orders of magnitude higher than that of the RNase H of RT (Post et al 1993); (ii) the PPT generated by the RNase H of RT is readily degraded by E. coli RNase HI (Luo et al 1990;Randolph & Champoux 1994;Guo et al 1995); (iii) the products generated by E. coli RNase HI range in size from two to nine nucleotides (Crouch 1990), whereas RNase H of RT yields RNAs of about 7-18 nucleotides (Oyama et al 1989;Schatz et al 1990;Fu & Taylor 1992). The size of the latter is thought to be due to the spacing between the polymerase and RNase H active sites on RT (Oyama et al 1989;Furfine & Reardon 1991;Kohlstaedt et al 1992;Jacobo-Molina et al 1993).…”

RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity

“…Both of these properties are absent in the isolated M-MuLV RNase H domain. Many studies indicate that the RNase H active sites of RTs are spatially located approximately 18 template bases away from the primer terminus in actively synthesizing complexes (11,14,17,20,29,37). We previously demonstrated that contacts between PPT hybrids and M-MuLV RT as far as 7 bp upstream of the cleavage site are critical for correctly positioning RNase H for the cleavage reaction that creates the plus-strand primer (36).…”

“…In a pH titration over this range, RT and RNH exhibited optimal activities at pH 8 whereas that of GST-RNH was optimal at pH 6.8 but not greatly reduced at pH 8.0 (data not shown). KCl was omitted from these reactions since the RNase H activity of RT is maximal in the absence of KCl (5,14) and the salt concentration does not affect the specificity of PBS primer removal for M-MuLV RT (38). During these studies, we found that the addition of 100 g of BSA per ml to the reaction mixtures significantly increased the amount of activity observed in the acid solubility assay without having any effect on the different specificity assays used for RNase H. When we substituted 32 P-labeled single-stranded RNA for the 32 P-labeled RNA-DNA hybrid in the nonspecific assay, the recombinant RNH and GST-RNH preparations contained negligible levels of RNase activity (data not shown).…”