Expansion of a (CTG)n segment within the 3'-untranslated region of the myotonic dystrophy protein kinase gene alters mRNA production. The inherent ability of RNA polymerase II to transcribe (CTG)17-255 tracts corresponding to DNA from normal, unstable, and affected individuals, and the normal (CGG)54 fragile X repeat tract, was analyzed using a synchronized in vitro transcription system. Core RNA polymerase II transcribed all repeat units irrespective of repeat length or orientation. However, approximately 50% of polymerases transiently halted transcription (with a half-life of approximately 10 +/- 1 s) within the first and second CTG repeat unit and a more transient barrier to elongation was observed roughly centered within repeats 6-9. Transcription within the remainder of the CTG tracts and within the CCG, CGG, and CAG tracts appeared uniform with average transcription rates of 170, 250, 300, and 410 nucleotides/min, respectively. These differences correlated with changes in the sequence-specific transient pausing pattern within the CNG repeat tracts; individual incorporation rates were slower after incorporation of pyrimidine residues. Unexpectedly, approximately 4% of the run-off transcripts were, depending on the repeat sequence, either 15 or 18 nucleotides longer than expected. However, these products were not produced by transcriptional slippage within the repeat tract.