USB1 pauses 5 nucleotides 3′ to an adenosine. (A) Wild-type and mutant 5′ fluorescently labeled oligoribonucleotides used in the assay. Mutations are highlighted in red. (B) USB1 reads the position of an upstream adenine nucleotide. The initial length of the oligos and the positions of the strong pause sites after incubation with USB1 are indicated. (C) USB1 pauses 5 nucleotides downstream of an adenine nucleotide independent of the total number of 3′ uridines.