To induce dissociation of the transcription elongation complex, a typical intrinsic terminator forms a G.C-rich hairpin structure upstream from a U-rich run of approximately eight nucleotides that define the transcript 3' end. Here, we have adapted the nucleotide analog interference mapping (NAIM) approach to identify the critical RNA atoms and functional groups of an intrinsic terminator during transcription with T7 RNA polymerase. The results show that discrete components within the lower half of the hairpin stem form transient termination-specific contacts with the RNA polymerase. Moreover, disruption of interactions with backbone components of the transcript region hybridized to the DNA template favors termination. Importantly, comparative NAIM of termination events occurring at consecutive positions revealed overlapping but distinct sets of functionally important residues. Altogether, the data identify a collection of RNA terminator components, interactions and spacing constraints that govern efficient transcript release. The results also suggest specific architectural rearrangements of the transcription complex that may participate in allosteric control of intrinsic transcription termination.