Circularized oligonucleotides, or coligos, were previously found to serve as RNA 
polymerase III (Pol III) templates in vitro and in human tissue culture cells. 
Here we randomized the 12-nucleotide larger loop (L-loop) of a well 
characterized coligo and found unexpectedly that in vitro transcription by 
FLAG-Pol III was not significantly affected. This observation allowed us to test 
the variable of coligo L-loop size separately from the variable of its sequence. 
Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of 
randomized sequence, and the smallest loop forced initiation to move into the 
stem region. To test further the need for any specific sequence we compared 
seven nucleotide L-loops composed of random, abasic and abasic-acyclic 
nucleotides, and all supported transcription by Pol III. Transcription of a 
series of coligos containing twelve contiguous randomized nucleotides placed at 
different locations within the coligo structure provided further evidence that 
the stem-loop junction structure is important for precise initiation. Nearly the 
same transcript pattern was formed in vitro by Pol III from yeast and human 
cells. Overall, these experiments support structure, rather than L-loop 
sequence, as the major determinant of coligo transcription initiation by Pol 
III.