The expression of the Escherichia coli K5 (group II) capsular polysaccharide requires the rfaH gene. By reverse transcriptase-polymerase chain reaction (RT-PCR) it was possible to demonstrate that RfaH increases the transcription of region 2 genes by readthrough transcription from the region 3 promoter. A mutation in the rfaH gene reduced this readthrough transcription from the region 3 promoter by 10-fold as measured by quantitative RT-PCR. The region 3 promoter was mapped to 741 bp 5' of the initiation codon of the kpsM gene. Deletion and insertion mutagenesis of the JUMPstart sequence, which is 28 bp 5' of kpsM and is conserved upstream of RfaH-regulated operons and other polysaccharide biosynthesis genes, confirmed that this sequence was required for expression of the K5 antigen and for the antitermination activity of RfaH. The JUMPstart sequence could cause RfaH-dependent antitermination at other Rho-dependent terminators, suggesting that the JUMPstart sequence may function in a manner analogous to a lambda nut site. On the basis of these results we propose a model by which RfaH regulates expression of E. coli group II capsule gene clusters by allowing readthrough transcription to proceed from region 3 into region 2 and that sequences within the JUMPstart sequence are essential for this process.