Activation of a Chimeric Rpb5/RpoH Subunit Using Library Selection

Abstract

Rpb5 is a general subunit of all eukaryotic RNA polymerases which consists of a N-terminal and a C-terminal domain. The corresponding archaeal subunit RpoH contains only the conserved C-terminal domain without any N-terminal extensions. A chimeric construct, termed rp5H, which encodes the N-terminal yeast domain and the C-terminal domain from Pyrococcus furiosus is unable to complement the lethal phenotype of a yeast rpb5 deletion strain (Δrpb5). By applying a random mutagenesis approach we found that the amino acid exchange E197K in the C-terminal domain of the chimeric Rp5H, either alone or with additional exchanges in the N-terminal domain, leads to heterospecific complementation of the growth deficiency of Δrpb5. Moreover, using a recently described genetic system for Pyrococcus we could demonstrate that the corresponding exchange E62K in the archaeal RpoH subunit alone without the eukaryotic N-terminal extension was stable, and growth experiments indicated no obvious impairment in vivo. In vitro transcription experiments with purified RNA polymerases showed an identical activity of the wild type and the mutant Pyrococcus RNA polymerase. A multiple alignment of RpoH sequences demonstrated that E62 is present in only a few archaeal species, whereas the great majority of sequences within archaea and eukarya contain a positively charged amino acid at this position. The crystal structures of the Sulfolobus and yeast RNA polymerases show that the positively charged arginine residues in subunits RpoH and Rpb5 most likely form salt bridges with negatively charged residues from subunit RpoK and Rpb1, respectively. A similar salt bridge might stabilize the interaction of Rp5H-E197K with a neighboring subunit of yeast RNA polymerase and thus lead to complementation of Δrpb5.

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

“…The resulting plasmid pMUR1 was sequenced and 1 µg of the linearized version was used for transformation of Pfu as described previously 78,79 . Transformants were enriched with 10 µM simvastatin in ½ SME-starch liquid medium at 85°C for 48 h, and pure cultures were isolated after plating the cells on solidified medium in the presence of 10 µM simvastatin.…”

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure

“…Rpb5 is located at the lower "jaw" of pol II (see above). The presence/absence of the N domain is, in fact, not critical for polymerase function insofar as the archaeal and yeast subunits can cross-complement (125). However, in common with the above-described theme, Rpb5 seems to mediate regulatory interactions (126)(127)(128).…”

“…The Rpb5 subunit of yeast is a 215-aa twodomain protein (125). However, the corresponding archaeal subunit is only 84 aa in length due to an entirely missing N-terminal domain.…”

Structural Insights into Nonspecific Binding of DNA by TrmBL2, an Archaeal Chromatin Protein

“…The primers are shown in Supplementary Table 1. The complete DNA fragment was transformed into P. furiosus as described previously [44,45]. The transformants were selected with the antibiotics simvastatin and pure cultures were isolated after plating the cells on solidified medium in the presence of 10 μM simvastatin.…”