The Role of the Lid Element in Transcription by E. coli RNA Polymerase

The recently described crystal structures of multi-subunit RNA polymerases (RNAPs) reveal a conserved loop-like feature called the lid. The lid projects from the clamp domain and contacts the flap, thereby enclosing the RNA transcript in RNAPʹs RNA-exit channel and forming the junction between the exit channel and the main channel, which holds the RNA:DNA hybrid. In the initiating form of bacterial RNAP (holoenzyme containing σ), the lid interacts with σ region 3 and encloses an extended linker between σ region 3 and σ region 4 in place of the RNA in the exit channel. During initiation, the lid may be important for holding open the transcription bubble and may help displace the RNA from the template DNA strand. To test these ideas, we constructed and characterized a mutant RNAP from which the lid element was deleted. Δlid RNAP exhibited dramatically reduced activity during initiation from −35-dependent and −35-independent promoters, verifying that the lid is important for stabilizing the open promoter complex during initiation. However, transcript elongation, RNA displacement, and, surprisingly, transcriptional termination all occurred normally in Δlid RNAP. Importantly, Δlid RNAP behaved differently from wild-type RNAP when transcribing single-stranded DNA templates where it synthesized long, persistent RNA:DNA hybrids, in contrast to efficient transcriptional arrest by wild-type RNAP.