Deletion of switch 3 results in an archaeal RNA polymerase that is defective in transcript elongation.
ABSTRACT 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.
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Article: Archaeal RNA polymerase.[show abstract] [hide abstract]
ABSTRACT: The recently solved X-ray crystal structures of archaeal RNA polymerase (RNAP) allow a structural comparison of the transcription machinery among all three domains of life. Archaeal transcription is very simple and all components, including the structures of general transcription factors and RNAP, are highly conserved in eukaryotes. Therefore, it could be a new model for the dissection of the eukaryotic transcription apparatus. The archaeal RNAP structure also provides a framework for addressing the functional role that Fe-S clusters play within the transcription machinery of archaea and eukaryotes. A comparison between bacterial and archaeal open complex models reveals likely key motifs of archaeal RNAP for DNA unwinding during the open complex formation.Current Opinion in Structural Biology 10/2009; 19(6):724-31. · 8.74 Impact Factor
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ABSTRACT: Evolutionarily related multisubunit RNA polymerases (RNAPs) facilitate gene transcription throughout the three domains of life. During the past seven years an increasing number of bacterial and eukaryotic RNAP structures have been solved; however, the archaeal enzyme remained elusive. Two reports from the Murakami and Cramer laboratories have now filled this gap in our knowledge and enable us to hypothesize about the evolution of the structure and function of RNAPs.Trends in Microbiology 07/2008; 16(6):247-50. · 8.43 Impact Factor
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ABSTRACT: Passage of E. coli RNA polymerase through an intrinsic transcription terminator, which encodes an RNA hairpin followed by a stretch of uridine residues, results in quick dissociation of the elongation complex. We show that folding of the hairpin disrupts the three upstream base pairs of the 8 bp RNA:DNA hybrid, a major stability determinant in the complex. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. During termination, the hairpin does not directly compete for base pairing with the 8 bp hybrid. Thus, melting of the hybrid seems to result from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Our results suggest that a similar mechanism disrupts elongation complexes of yeast RNA polymerase II in vitro.Molecular Cell 12/2002; 10(5):1151-62. · 15.28 Impact Factor