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.

One common bacterial type of transcriptional slippageprone sequence involves 9 or more A's or T's (28) ; other repeats have also been analyzed (29) . Dissociation of the nascent RNA from its template hybrid complement allows realigned pairing in either direction. A well known Paramyxovirus heteropolymeric slippage motif is composed of A's followed by G's with the identity of the mis-paired base in the new re-aligned hybrid being important in determining slippage directionality (30, 31) . Nearly all work has focused on slippage involving a linear (unstructured) template. However, there is evidence that a protein roadblock or template structure ahead of a DNA-dependent RNA polymerase transcribing a slippage motif can stimulate realignment (2, 32, 33) . Also there is one report of roadblock-mediated RT slippage where a polymerase bypasses an RNA-structure forming sequence prior to resumption of synthesis (34) . The present work does not explore RT generation of product lacking sequence complementary to template sequence present in RNA structure.