Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

In the absence of template RNA structure, when RT polymerase transcribes a 'slippery' sequence the cDNA:RNA hybrid is prone to realign in either direction. cDNA backward realignment is at a lower level than when a template structure is at the leading edge of the polymerase ( Figure 6 , from C to G). cDNA forward realignment involves RT polymerase in the post-translocation state being transformed to the pre-translocation state ( Figure 6 , from B to F). Evidence for this assertion comes from several aspects of the results. Firstly, high relative substrate concentration specified by the motif base inhibits slippage that generates product lacking a base compared to the template. This is perhaps due to pairing of substrate base with the template base at the catalytic center as it would prevent pairing of the forward realigned cDNA 3 end base ( Figure 6B) . Secondly, the finding that relatively higher concentration of the substrate, specified by the template base 5 adjacent to the motif, strongly stimulates base omission, is explicable by re- Figure 6 . Model of RNA template structure influence on RT slippage. RT enzyme (open black rectangle) with the RNA template (red) and the nascent cDNA (blue). The polymerase and RNase H catalytic centers are pink and green rectangles respectively. The two 5 bases of the RNA slippage motif are indicated by green closed circles and the base 5 adjacent to the motif is indicated by a brown closed circle. Their corresponding cognate substrate is indicated with green and brown closed squares respectively. Inhibition and stimulation effect are indicated by -and + symbols. Standard RT transcription (A-E). Forward realignment-mediated base omission (B-F) occurs from a polymerase Post-translocation state in the absence of the cognate substrate in the catalytic center, and following polymerase forward translocation (F-D) is productively locked by incorporation of the substrate specified by the next template base (D-E). Backward realignment-mediated base addition (C-G) occurs from a polymerase Pre-translocation state stimulated by the formation of the template structure (H), and is productively locked by incorporation of the cognate substrate (G-C). alignment locking following this template base locating in the catalytic center ( Figure 6 from F to D). Consistent with this, availability of the base 5 adjacent to the RNA motif is crucial for base omission since its sequestration in template stem pairing (intra-template or antisense pairing) ( Figure 6H ) inhibits base omission ( Figure 6 , from F to D). Accordingly, a requisite for productive forward realignment is that prior to its occurrence, the RT polymerase is in a posttranslocation state with the 3 end of the cDNA being base paired to the template base second from the 5 end of the RNA motif ( Figure 6B ). Even though substrate base pairing serves to lock realigned hybrid pairing, the potential for reversal of realignments is indicated by the strong effect of relative dNTP concentration on modulating slippage directionality. This implies that substrate pairing is slower than realigned hybrid formation.