In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support 
enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites 
from different species appear to exhibit differential intrinsic transcript 
cleavage efficiency and have likely evolved to allow fine-tuning of the 
transcription process. Here we show that a single amino-acid substitution in the 
trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of 
intrinsic cleavage activity where the substituted tyrosine appears to 
participate in acid-base chemistry at alkaline pH for both intrinsic cleavage 
and nucleotidyl transfer. We extensively characterize this TL substitution for 
each of these reactions by examining the responses RNAP II enzymes to catalytic 
metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of 
the first phosphodiester bond formation by RNAP II requires wild type TL 
function and that H1085Y substitution within the TL compromises or alters RNAP 
II responsiveness to both TFIIB and TFIIF. Finally, Mn(2+) stimulation of H1085Y 
RNAP II reveals possible allosteric effects of TFIIB on the active center and 
cooperation between TFIIB and TFIIF.