During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet it must access the nucleosomal DNA. It has been proposed that multiple RNAPs might increase transcription efficiency through nucleosomal DNA. Here we have quantitatively investigated this hypothesis by using E. coli RNAP as a model system and directly monitoring its location on the DNA via a single molecule DNA unzipping technique. When a single RNAP encountered a nucleosome, it paused with a distinctive 10-bp periodicity and was backtracked by an average distance of ~10-15 bp. When two RNAPs were elongating in close proximity, the trailing RNAP exerted an assisting force on the leading RNAP, reducing its backtracking and enhancing its transcription through a nucleosome ~5-fold. Taken together, our data indicate that histone-DNA interactions within a nucleosome dictate RNAP pausing behavior, and that alleviation of nucleosome-induced backtracking by multiple polymerases is a likely mechanism for overcoming the nucleosomal barrier in vivo.