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-nucleotide (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 template terminal complexes produce these RNAs and that transcript extension is a general and salt-sensitive (250 mM) feature of run-off transcription. On 5'-overhung templates the extended run-off transcripts appear to be retained within an RNA-DNA-enzyme ternary complex, and SII facilitates resumption of transcript elongation via a dinucleotide truncation intermediate. Moreover, on one of the 5-overhung templates, the initially extended complexes spontaneously resumed transcript extension and were uniquely resistant to salt (250 mM) challenge. However, SII did not facilitate this long distance extension on all template ends. Run-off transcripts on a blunt-ended template were initially extended by 2-11 nt (roughly in 2-nt increments); SII addition either before or after extension resulted in the accumulation of a 4-5-nt extension product. Based on these findings, we propose that the initial and continuously extended RNAs reflect intermediates and successful completion of template end-to-end transposition (template switching) by RNA polymerase II, respectively. Both the template end sequence and structure influenced the success of such an event.
Department of Obstetrics and Gynecology, Sealy Center for Molecular Science, the University of Texas Medical Branch, Galveston, Texas 77555-1062, USA. Mizban@utmb.edu
M G
MG
Izban
M A
MA
Parsons
R R
RR
Sinden
D004247:DNA / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000737:chemistry; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013698:Templates, Genetic
D012333:RNA, Messenger; D011189:Potassium Chloride; D004247:DNA; D012319:RNA Polymerase II; D015246:Deoxyribonuclease EcoRI