SignificancePausing by RNA polymerase is essential for transcription in all domains of life. Little is known about how pausing is regulated bytrans-acting factors andcis-acting signals in DNA/nascent RNA in vivo. Employing an advanced version of (nascent elongating transcript sequencing) NET-seq (RNET-seq), we found that the universally conserved transcription elongation factor NusG is a robust suppressor of backtracked and nonbacktracked pauses inEscherichia coli. Our in vivo work revealed a similarity of NusG to Spt5, its archaeal and eukaryotic homolog, which regulates the elongation rate of many eukaryotic genes.