Significance During transcription elongation at saturating nucleotide concentrations, RNA polymerase (RNAP) performs ∼50 nucleotide-addition cycles every second. The RNAP active center contains a structural element, termed the trigger loop (TL), that has been suggested, but not previously shown, to open to allow a nucleotide to enter and then to close to hold the nucleotide in each nucleotide-addition cycle. Here, using single-molecule fluorescence spectroscopy to monitor distances between a probe incorporated into the TL and a probe incorporated elsewhere in the transcription elongation complex, we show that TL closing and opening occur in solution, define time scales and functional roles of TL closing and opening, and, most crucially, demonstrate that one cycle of TL closing and opening occurs in each nucleotide-addition cycle.