Deletion of switch 3 results in an archaeal RNA polymerase that is defective in transcript elongation.
Switch 3 is a polypeptide loop conserved in all multisubunit DNA-dependent RNA polymerases (RNAPs) that extends into the main cleft of the RNAP and contacts each base in a nascent transcript as that base is released from the internal DNA-RNA hybrid. Plasmids have been constructed and transformed into Thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal RNAP subunit RpoB with an N-terminal His(6) tag and the Switch 3 loop either intact (wild-type) or deleted (DeltaS3). RNAPs containing these plasmid-encoded RpoB subunits were purified, and, in vitro, the absence of Switch 3 had no negative effects on transcription initiation or elongation complex stability but reduced the rate of transcript elongation. The defect in elongation occurred at every template position and increased the sensitivity of the archaeal RNAP to intrinsic termination. Comparing these properties and those reported for a bacterial RNAP lacking Switch 3 argues that this loop functions differently in the RNAPs from the two prokaryotic domains. The close structural homology of archaeal and eukaryotic RNAPs would predict that eukaryotic Switch 3 loops likely conform to the archaeal rather than bacterial functional paradigm.
Department of Microbiology, Ohio State University, Columbus, OH 43210, USA. santangelo.11@osu.edu
Thomas J
TJ
Santangelo
John N
JN
Reeve
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D017353:Gene Deletion; D006493:Heparin / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D019642:RNA, Archaeal / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D019712:Thermococcus / Q000201:enzymology / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D019642:RNA, Archaeal; D014157:Transcription Factors; D012313:RNA; D006493:Heparin; D004247:DNA; D012321:DNA-Directed RNA Polymerases