TEMPLATE END-TO-END TRANSPOSITION BY RNA-POLYMERASE-II

On 5'-template strand protruding templates, promoter-initiated run-off transcription by RNA polymerase II generates discrete, 15-16-nucleoti de (nt) longer than expected products whose production is abrogated by elongation factor SII (Parsons, M. A., Sinden, R. R., and Izban, M. G . (1998) J. Biol. Chem. 273, 26998-27008). We demonstrate that templat e terminal complexes produce these RNAs and that transcript extension is a general and salt-sensitive (250 mM) feature of run-off transcript ion. On 5'-overhung templates the extended run-off transcripts appear to be retained within an RNA-DNA-enzyme ternary complex, and SII facil itates resumption of transcript elongation via a dinucleotide truncati on intermediate. Moreover, on one of the 5-overhung templates, the ini tially extended complexes spontaneously resumed transcript extension a nd were uniquely resistant to salt (250 mM) challenge. However, SII di d not facilitate this long distance extension on all template ends. Ru n-off transcripts on a blunt-ended template were initially extended by 2-11 nt (roughly in 2-nt increments); SII addition either before or a fter extension resulted in the accumulation of a 4-5-nt extension prod uct. Based on these findings, we propose that the initial and continuo usly extended RNAs reflect intermediates and successful completion of template end-to-end transposition (template switching) by RNA polymera se II, respectively. Both the template end sequence and structure infl uenced the success of such an event.