Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the 
RNA transcript can thread back onto the template DNA strand, resulting in an R 
loop. Previously, we showed that initiation of R-loop formation at an R-loop 
initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro 
system with T7 RNA polymerase, we show that increased distance between the 
promoter and the R-loop-supporting G-rich region reduces R-loop formation. When 
the G-rich portion of the RNA transcript is downstream from the 5' end of the 
transcript, the ability of this portion of the transcript to anneal to the 
template DNA strand is reduced. When we nucleolytically resect the beginning of 
the transcript, R-loop formation increases because the G-rich portion of the RNA 
is now closer to the 5' end of the transcript. Short G-clustered regions can act 
as RIZs and reduce the distance-induced suppression of R-loop formation. 
Supercoiled DNA is known to favor transient separation of the two DNA strands, 
and we find that this favors R-loop formation even in non-G-rich regions. Most 
strikingly, a nick can serve as a strong RIZ, even in regions with no G 
richness. This has important implications for class switch recombination and 
somatic hypermutation and possibly for other biological processes in transcribed 
regions.