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 RNAP's 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 sigma), the lid interacts with sigma region 3 and encloses an extended linker between sigma region 3 and sigma 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. Deltalid 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 Deltalid RNAP. Importantly, Deltalid 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.
Toulokhonov
Innokenti
I
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA. itoulokhonov@wisc.edu
Landick
Robert
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D017353:Gene Deletion; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011196:Potassium Permanganate / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D016914:Ribonuclease H / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D012808:Sigma Factor; D011196:Potassium Permanganate; C081775:RNA polymerase sigma 70; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; D006163:Ribonuclease T1