Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive

Abstract

All archaeal genomes encode RNA polymerase (RNAP) subunits E and F that share a common ancestry with the eukaryotic RNAP subunits A43 and A14 (Pol I), Rpb7 and Rpb4 (Pol II), and C25 and C17 (Pol III). By gene replacement, we have isolated archaeal mutants of Thermococcus kodakarensis with the subunit F-encoding gene (rpoF) deleted, but we were unable to isolate mutants lacking the subunit E-encoding gene (rpoE). Wild-type T. kodakarensis grows at temperatures ranging from 60 degrees C to 100 degrees C, optimally at 85 degrees C, and the DeltarpoF cells grew at the same rate as wild type at 70 degrees C, but much slower and to lower cell densities at 85 degrees C. The abundance of a chaperonin subunit, CpkB, was much reduced in the DeltarpoF strain growing at 85 degrees C and increased expression of cpkB, rpoF or rpoE integrated at a remote site in the genome, using a nutritionally regulated promoter, improved the growth of DeltarpoF cells. RNAP preparations purified from DeltarpoF cells lacked subunit F and also subunit E and a transcription factor TFE that co-purifies with RNAP from wild-type cells, but in vitro, this mutant RNAP exhibited no discernible differences from wild-type RNAP in promoter-dependent transcription, abortive transcript synthesis, transcript elongation or termination.

Fig. 1. Construction of plasmids designed to delete TK0901 (rpoF) and TK1699 (rpoE), and confirmation of deletion (ΔTK0901; ΔrpoF) inT. kodakarensis KU2F

(A) Plasmids pUDRF, pUDRE1 and pUDRE2 were constructed with pyrF (TK2297) flanked by ~0.7 to 1 kbp of the T. kodakarensis genome as illustrated. These plasmids were used to transform KU216 (ΔpyrF). The pairs of oligonucleotides (F1 and F2; F3 and F4; sequences in Supplementary Table 1) used to PCR amplify genomic DNA from pUDRF-generated uracil-independent transformants are indicated. (B) Electrophoresis of DNA molecules PCR amplified from T. kodakarensis KU216 (lanes 1 and 3) and KU2F (lanes 2 and 4) using primer pairs F1 and F2 (lanes 1 and 2), or F3 and F4 (lanes 3 and 4). The DNA molecules amplified using primers F1 and F2 from T. kodakarensis KU216 and KU2F were, as predicted, 1.8 and 2.1 kbp, respectively. Lane S contained size standards. (B) Western blot analysis of the polypeptides present in aliquots of lysates of T. kodakarensis KU216, KU2F and KUWFF separated by SDS-PAGE. The control lane (C) contained a sample of the purified E plus F subunits used to generate the rabbit anti-subunit E and anti-subunit F antibodies. Total 10 ng proteins were applied to each lane of the gel.

Fig. 2. Growth ofT. kodakarensis KU216 (●) and KU2F(○) cultures at 70 and 85 °C

Aliquots of overnight cultures were diluted into ASW-YT-S⁰ medium containing 1% (w/v) sodium pyruvate and the cultures were then incubated (A) at 70 °C and (B) at 85 °C.

Fig. 3. Western blot analysis of CpkA and CpkB synthesis at 70 and 85 °C

The polypeptides in aliquots (5 µg) of lysates of T. kodakarensis KU216 and KU2F cells grown at 70 or 85 °C were separated by SDS-PAGE. CpkA and CpkB were detected by western blot analysis using antibodies specific for T. kodakarensis CpkA (top) or CpkB (bottom).

Fig. 4. Structure of the selection-expression cassettes and integration into the TK1765-TK1766 region of theT. kodakarensis genome

(A) The three plasmids constructed, pCTB, pCTE and pCTF (Table 1) contained the 5' and 3' regions of TK1765 (chiA), the intergenic region downstream of TK1765 with the t_chiA transcription terminator and TK1766. The selection-expression cassettes cloned between the 5' and 3' regions of TK1765 contained the selectable marker gene (trpE; TK0254), a transcription terminator (t_gdh), the regulated P_TK2164 promoter (P_FBPase), and either TK2303 (cpkB; pCTB), TK1699 (rpoE; pCTE) or TK0901 (rpoF; pCTF). These plasmids were used to transform T. kodakarensis KUWF (ΔrpoF∷pyrF; ΔtrpE). Integration of the expression cassettes into the TK1765-TK1766 region of the KUWF genome was confirmed by PCR amplification of genomic DNAs using primer sets including: 1) ChF and ChR; and 2) ChR and FF1, EF1 or BF1 (sequences in Supplementary Table 1) that hybridized at the locations indicated black arrows. (B) Electrophoresis of DNA molecules PCR amplified from T. kodakarensis KUWF, KUWFF, KUWFE and KUWFB using primer pairs FF1 and ChR (left panel), EF1 and ChR (middle panel), or BF1 and ChR (right panel). The DNA molecules amplified using these primer pairs were indicated by arrows. Lane M contained size standards.

Fig. 5. Media-dependent growth ofT. kodakarensis strains at 70 and 85°C

Aliquots of overnight cultures of T. kodakarensis KUW1 (ΔpyrF; ΔtrpE), KUWF (ΔrpoF∷pyrF; ΔtrpE) and KUWFF (ΔrpoF∷pyrF; ΔtrpE; ΔchiA∷trpE-P_TK2164-rpoF) were diluted into (A) ASW-YT-S⁰-Pyr medium (gluconeogenic growth condition; P_TK2164 active) or (B) ASW-YT-Mdx medium (glycolytic growth condition; P_TK2164 inactive) and incubated for 16 h at 70°C or 85°C. The OD₆₀₀ reached after the 16 h incubation period is listed below the culture.

Fig. 6. Partial suppression of the temperature-sensitive growth phenotype of KUWF (ΔrpoF) by rpoE and cpkB expression

Cultures of T. kodakarensis KUW1 (ΔpyrF; ΔtrpE;●), KUWF (ΔrpoF∷pyrF; ΔtrpE;○), KUWFF (ΔrpoF∷pyrF; ΔtrpE; ΔchiA∷trpE-P_TK2164-rpoF; X), KUWFE (ΔrpoF∷pyrF; ΔtrpE; ΔchiA∷trpE-P_TK2164-rpoE; ∆) and KUWFB (ΔrpoF∷pyrF; ΔtrpE; ΔchiA∷trpE-P_TK2164-cpkB, ▲) were grown at 85°C in ASW-YT-S⁰-Pyr (gluconeogenic growth condition; P_fbp active). Turbidity measurements were made using a Klett spectrophotomer (100 Klett units was equivalent to an OD₆₆₀ of 0.65).

Fig. 7. Ni²⁺-affinity purification andin vitro transcription byT. kodakarensis RNAPs

(A) Commassie blue staining after SDS-PAGE of aliquots of RNAP (7.5 µg) purified from T. kodakarensis TS413 and KUWLFB. The control lane (S) contained size standards (kDa). RNAP subunits E (RpoE) and F (RpoF) and TFE are present only in RNAP preparations from T. kodakarensis TS413. The L subunits in the RNAPs from T. kodakarensis TS413 and KUWLFB have HA+his₆ and his₆ C-terminal extensions, respectively, that result in different electrophoretic mobilities. (B) Comparison of transcripts synthesized in vitro directed by the constitutive archaeal promoters P_gdh or P_hmtB in reaction mixtures that contained either TFB1 (TK1280) or TFB2 (TK2287) and RNAP from TS413 or KUWLFB (Santangelo et al. 2006; 2007). Aliquots of RNAP (40 nM) were incubated with 80 nM TBP, 80 nM TFB1 (1) or TFB2 (2) and 10 nM of a template for 5 min at 85°C, and transcription then initiated by adding 500 µM ATP, CTP and GTP,50 µM UTP plus 0.5 µCi of [³²P]-α-UTP. The [³²P]-labeled transcripts synthesized during 5 min incubation at 85°C were precipitated, separated by electrophoresis and visualized using a Storm 840 phosphorimager. The image shows the relative amounts of the full-length run-off transcripts synthesized from the two promoters from initiation complexes containing either TFB1 or TFB2.

Fig. 8. Temperature and template-dependent transcript synthesis in vitro by T. kodakarensis RNAPs

(A) Electrophoretic separation of the transcripts synthesized by RNAP from T. kodakarensis TS413 or KUWLFB from template T452 (sequence in Supplementary Table 2) during incubation for 5 min at temperatures increasing from 50 to 55, 60, 65, 70, 75, 80 and 85 °C. Transcription was initiated from P_hmtB and the transcripts synthesized were labeled at their 5'-terminus by [³²P]-ApC incorporation. (B) Electrophoretic separation of the transcripts synthesized by RNAP from T. kodakarensis TS413 or KUWLFB from templates T452, T443 or T468 (sequence in Supplementary Table 2) during incubation for 5 min at 85 °C. These templates all contain the same P_hmtB promoter and transcription was initiated at the same location. The downstream transcribed DNA in T452 contained an archaeal protein coding sequence (T452), whereas T443 and T468 contained archaeal intergenic sequences. All transcripts synthesized were labeled at their 5'-terminus by [³²P]-ApC incorporation. As indicated, additional investigations confirmed the identity of transcripts that resulted from abortive initiation, template-directed intrinsic termination and template run-off (TJS, unpublished results).