Table 1.
Strains and plasmids used in this study.
Figure 1.
Superimposition of the structure and sequence alignment of Rpb5 from S. cerevisiae and RpoH from P. furiosus.
A: The N-terminal part of Rpb5 is shown in orange and the C-terminal part in red [1]. The RpoH model in green was obtained using the program Modeller in combination with the solved RpoH structures of Methanocaldococcus jannaschii, Methanobacterium thermoautotrophicus and S. solfataricus [2], [9], [44]. For superpositioning the program DaliLite (www.ebi.ac.uk/Tools/structure/dalilite) was used. The amino acids at the N-terminus of RpoH which do not fit with the Rpb5 structure are shown in grey. The N-termini and the C-termini of Rpb5 and RpoH are labeled. B: The Rpb5 sequence of yeast and the RpoH sequence of P. furiosus were analyzed using a webserver of the program ClustalW2 (www.ebi.ac.uk/Tools/msa/clustalw2). Conserved amino acids are shown in red colour.
Figure 2.
Complementation of the Δrpb5 strain.
A: Schematic drawing of the constructs used for complementation. Plasmid pRS423_rpb5 contains the wild type sequence as a positive control. The chimeric construct pRS423_rp5H codes for the N-terminal region of Rpb5 and the C-terminal region of RpoH from P. furiosus. Transformants were cultivated on synthetic dropout plates (without histidine) containing FOA and incubated at 30°C for 10 days. B: Amino acid sequence of the chimeric Rp5H construct without the Flag tag at the N-terminus. The N-terminal domain of Rpb5 is shadowed in grey followed by the C-terminal RpoH domain. The sequences excluded from random mutagenesis are boxed.
Figure 3.
A single exchange within a chimeric Rp5H subunit can complement the loss of Rpb5 in yeast.
A: Overview about the analyzed mutants and data on growth behavior. The mutants were grown in liquid culture in synthetic drop-out medium without histidine at 24°C. The maximum OD600 and the time needed to grow to half of the maximum OD600 are indicated in the table. B: Growth comparison of Δrpb5 transformed with the positive control pRS423_rpb5 and the pRS423_rp5H (M75K + E197K) mutant on synthetic dropout plates (without histidine) containing FOA at 24°C and 37°C after five days of incubation.
Figure 4.
Schematic drawing of the plasmids used for the construction of the Pyrococcus mutants.
The plasmids were assembled by overlapping PCR. Plasmid pMUR27 contains the wild type sequence of RpoH and plasmid pMUR28 comprises RpoH with the single exchange E62K. Plasmids pMUR54 and pMUR43 contain the chimeric constructs. SimR is an additional copy of the hydroxymethylglutaryl CoA reductase from Thermococcus kodakarensis (TK0914) and provides resistance against simvastatin. For purification, a His6 and a Strep tag is available at the N-terminal region of all constructs. At the right side of the figure the stability of the corresponding Pyrococcus mutants is indicated.
Figure 5.
Purification and functional analysis of the RNAP.
A: Silver-stained 10 to 20% gradient Tris Tricine SDS gel. Each lane contains 3 µg of purified RNAP. The analyzed RNAP fractions were purified using a His6 tag at subunit D (lane 1) or subunit H (lanes 2 and 3). B: In vitro transcription with the purified RNAP fractions. Identical amounts of RNAPs were used to transcribe the gdh template in the presence of the archaeal transcription factors TBP and TFB. The transcription assays were performed as described previously [36].
Figure 6.
Sequence alignment and structural analysis of RpoH.
A: Part of a multiple alignment of several RpoH sequences. For the alignment a webserver of the program ClustalW2 was used (www.ebi.ac.uk/Tools/msa/clustalw2). Conserved amino acids are shown in red and the positions corresponding to E62 of P. furiosus are shadowed in grey. B: Overall structure of the archaeal RNAP from S. shibatae (ID code in the Protein Data Bank: 2WAQ). Subunits A″, H and K are shown in space filling mode with different colors. Residue R64 (E62 in P. furiosus) of subunit H is shown in blue, and the putative salt bridge interaction partner D12 of subunit K is colored in red. In the close-up the distances between the nitrogen atoms of the arginine side chain and the oxygen atoms of the glutamate side chain are indicated in Å. The figures were drawn using the program PyMOL (www.pymol.org). C: Close-up of a putative salt bridge between K197 of Rpb5 and D1013 of RpbA from S. cerevisiae (ID code in the Protein Data Bank: 4BBR). The distances between the nitrogen atom of the lysine side chain and the oxygen atoms of the glutamate side chain are indicated in Å.