Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G(-10)Y(-1)G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.