1. A Recombinant RNA Polymerase II-like Enzyme Capable of Promoter-Specific Transcription 
Finn Werner, Robert O.J Weinzierl 
Molecular Cell 
Volume 10, Issue 3, Pages (September 2002)
DOI: /S (02)

2. Figure 1 Structural Similarities between Archaeal and Eukaryotic RNAPs
(A) Schematic representation of the subunits of eukaryotic (S. cerevisiae) RNA polymerase II. Protein-protein interactions determined from the X-ray structure are indicated by solid and dotted lines to highlight the extent of the interactions (after Cramer et al., 2000).
(B) Schematic representation of the subunits of archaeal RNA polymerase. Protein-protein interactions are indicated by solid and dotted lines similar to (A) and were in most cases experimentally verified by the assembly studies described here.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
Expression, Purification, and Assembly of Recombinant Archaeal RNAP Subunits
(A) The purification/assembly strategies for recombinant versions of M. jannaschii RNAP subunits are outlined schematically.
(B) Purified recombinant subunits used for in vitro assembly. Aliquots of purified subunits were analyzed by SDS gel electrophoresis (stained with Coomassie blue).
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3 Assembly of M. jannaschii RNAP from Recombinant Subunits
(A) Fractions of a Superose 6 size exclusion chromatographic analysis of the renatured assembly reaction analyzed by SDS gel electrophoresis (stained with silver). Fractions 22 to 28 contain mostly high molecular weight aggregates of structurally heterogeneous and denatured subunits. Fractions 30 to 38 contain predominantly assembled RNAP. Note the near-stoichiometric presence of the various subunits. Fractions 40 to 42 contain the bulk of small subunits that were added in excess to promote RNAP assembly. Further purification of the assembled enzyme (fractions 30 to 38) on MonoQ revealed that the subunit stoichiometry of the recombinant enzyme was stable and invariant (data not shown).
(B) Presence of certain subunits not identified in (A) in the assembled RNAP as detected by Western blotting with specific antibodies. Note that excess H and K subunits are mostly present as high molecular weight aggregates.
(C) Nonspecific transcription assay. Identical aliquots of individual fractions were assayed for the presence of RNAP activity. The peak of transcriptional activity (fractions 34 to 36) corresponds to the peak of correctly assembled RNAP identified in (A). The α-32P-rUTP incorporation is shown on an arbitrary scale.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4 Functional Characterization of the Recombinant Archaeal RNAP
(A) The incorporation of α-32P-rUTP was measured in parallel in nonspecific transcription assays. The recombinant enzyme displays clear optima for reaction temperature and ion concentration in the reaction mixtures.
(B) The incorporation of α-32P-rUTP was measured in parallel in nonspecific transcription assays. Various controls are shown to illustrate the specificity of the assay and to study the functional properties of the enzyme. The differences in assay conditions are indicated along the x axis.
(C) Assembly of the TBP/TFB complex on the SSV T6 promoter. An electrophoretic mobility shift assay was used to monitor the assembly of recombinant M. jannaschii TBP and TFB on an oligonucleotide containing the SSV T6 promoter sequence.
(D) Promoter-directed transcription assays. The appearance of a specifically initiated transcript (arrow) is entirely dependent on the combined presence of TBP, TFB, and RNAP.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 Functional Role of Various Subunits
(A) Nonspecific transcription assays. RNAPs containing the specified subunit combinations were assembled strictly in parallel. After incubation for 10 min at 70°C (to remove nonspecific aggregates) the protein concentrations of the remaining soluble fractions were quantitated and used to compare the specific activities of the assemblies relative to the wild-type enzyme.
(B) Role of subunit N in stabilizing A′- and B′-containing assemblies. The soluble assemblies resulting from the complete set of subunits or lacking either subunits N (ΔN), P (ΔP), or both (ΔNP) were analyzed by SDS gel electrophoresis (small subunits not shown) after exposure to 70°C. Subunit B′ (and to a certain extent, A′) is only inefficiently retained in the soluble assembly in the absence of subunit N, whereas the absence of subunit P has no detectable effect on the stoichiometry of A′ and B′.
(C) Promoter-directed transcription assays. The assemblies lacking subunits E–F, H, or K are still capable of initiating transcription at the correct start site of the SSV6 promoter.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6 Site-Directed Mutagenesis of the Active Site
(A) The two metal catalytic mechanism; two Mg2+ ions are specifically coordinated by the carboxylate residues shown in red. The names of the carboxylates shown reflect the positions of the residues as found in the M. jannaschii A′ (aspartic acid residues D466, D468, and D470) and B′ polypeptide chain (glutamic acid residue E224 and aspartic acid residue D225) (after Steitz, 1998).
(B) Sequence homology/identity in the Mg2+ binding sites in RNAPs derived from all three evolutionary domains. The sequences of metal A and metal B motifs from archaea (mj; Methanococcus jannaschii), bacteria (ec; Escherichia coli), and eukarya (sc; Saccharomyces cerevisiae) are shown in alignment. The absolutely conserved carboxylates required for Mg2+ chelation are highlighted in red. Note the high degree of sequence conservation in the flanking sequences, especially in the metal A motif.
(C) Nonspecific transcription assays. RNAPs containing either wild-type or mutated large subunits were assembled under comparable conditions in parallel and standardized according to protein concentration. The mutations are as indicated and use the same nomenclature as that shown in (A).
(D) Promoter-directed transcription assays. Identical quantities of assembled wild-type and mutant RNAPs (mutations as indicated) were tested for specifically initiated transcripts in the presence of two different rNTP concentrations by primer-extension.
Molecular Cell  , DOI: ( /S (02) )