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.
SourceAvailable from: Thomas J Santangelo[Show abstract] [Hide abstract]
ABSTRACT: Archaeal histones wrap DNA into complexes, designated archaeal nucleosomes that resemble the tetrasome core of a eukaryotic nucleosome. Therefore, all DNA interactions in vivo in Thermococcus kodakarensis, the most genetically versatile model species for archaeal research, must occur in the context of a histone-bound chromosome. Here we report the construction and properties of T. kodakarensis strains that have TK1413 or TK2289 deleted, the genes that encode HTkA and HTkB, respectively, the two archaeal histones present in this Archaeon. All attempts to generate a strain with both TK1413 and TK2289 deleted were unsuccessful arguing that histone-mediated event(s) in T. kodakarensis are essential. The HTkA and HTkB amino acid sequences are 84% identical (56/67) and 94% (63/67) similar but despite this homology, and their apparent redundancy in terms of supporting viability, the absence of HTkA and HTkB resulted in differences in growth and in quantitative and qualitative differences in genome transcription. A most surprising result was that deletion of TK1413 (ΔhtkA) resulted in a T. kodakarensis strain that is no longer amenable to transformation whereas deletion of TK2289 (ΔhtkB) had no detrimental effects on transformation. Potential roles for the archaeal histones in regulating gene expression, and for HTkA in DNA uptake and recombination are discussed.Journal of bacteriology 10/2012; 194(24). DOI:10.1128/JB.01523-12 · 2.69 Impact Factor
Article: Genetic Techniques for the Archaea.[Show abstract] [Hide abstract]
ABSTRACT: Genetic techniques for the Archaea have undergone a rapid expansion in complexity, resulting in increased exploration of the role of Archaea in the environment and detailed analyses of the molecular physiology and information-processing systems in the third domain of life. Complementary gains in describing the ever-increasing diversity of archaeal organisms have allowed these techniques to be leveraged in new and imaginative ways to elucidate shared and unique aspects of archaeal diversity and metabolism. In this review, we introduce the four archaeal clades for which advanced genetic techniques are available-the methanogens, halophiles, Sulfolobales, and Thermococcales-with the aim of providing an overall profile of the advantages and disadvantages of working within each clade, as essentially all of the genetically accessible archaeal organisms require unique culturing techniques that present real challenges. We discuss the full repertoire of techniques possible within these clades while highlighting the recent advances that have been made by taking advantage of the most prominent techniques and approaches. Expected final online publication date for the Annual Review of Genetics Volume 47 is November 23, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual Review of Genetics 09/2013; DOI:10.1146/annurev-genet-111212-133225 · 18.12 Impact Factor
Chemical Reviews 09/2013; 113(11). DOI:10.1021/cr4002325 · 45.66 Impact Factor