Transcription by RNA polymerase can stimulate negative DNA supercoiling in Escherichia coli topA strains. This phenomenon has been explained by a "twin-supercoiled-domain" model of transcription in which positive DNA supercoils are generated in front of a translocating RNA polymerase and negative supercoils behind it. However, since there is lack of a specific system to study the factors governing this biologically important process, the parameters regulating transcription-coupled DNA supercoiling (TCDS) in E.coli still remain elusive. Here, we describe our efforts to study TCDS in E.coli using a newly developed system. This system consists of a topA strain, VS111(DE3) or DM800(DE3), in which a lambdaDE3 prophage containing a T7 RNA polymerase gene under the control of lacUV5 promoter has been integrated into the cell chromosome, along with a set of plasmids producing RNA transcripts of various lengths by T7 RNA polymerase. Using this system, we found that transcription by T7 RNA polymerase strikingly induced the formation of hypernegatively supercoiled plasmid DNA. We also discovered, for the first time, that TCDS was dependent on the length of RNA transcripts in vivo, precisely predicted by the twin-supercoiled-domain model of transcription. Furthermore, our results demonstrated that hypernegative supercoiling of plasmid DNA by T7 RNA polymerase did not require anchoring of DNA to the bacterial cytoplasmic membrane. These results indicate that a transcribing RNA polymerase alone is sufficient to cause a change in local DNA superhelicity, which can have a powerful impact on the conformation and function of critical DNA sequence elements such as promoters and DNA replication origins.