Methanobacterium thermoautotrophicum RNA polymerase and transcription in vitro

Abstract

RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum DeltaH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.

FIG. 1

Identities of M. thermoautotrophicum ΔH RNAP subunits silver stained after separation by tricine-SDS-PAGE. Stetter et al. (24) identified eight subunits (designated by the letters O and A through G) in RNAP preparations from M. thermoautotrophicum Winter. Based on estimated sizes and very similar patterns of SDS-PAGE resolution, O and A through F appear to have been homologs of the M. thermoautotrophicum ΔH subunits here designated A′, B′, B", A", D, E′, and F, and subunit G was a mixture that contained the four polypeptides designated subunits H, L, K, and P. With the exception of subunits F and P, the M. thermoautotrophicum ΔH subunits are homologs of subunits identified previously for Sulfolobus acidocaldarius RNAP (14, 15). M. thermoautotrophicum ΔH RNAP preparations did not contain a homolog of the S. acidocaldarius subunit G, and the MTH0265 and MTH0040 gene products, predicted to be RNAP subunits E" (6.7 kDa) and N (6.4 kDa) (23), were not detected, although their presence may have been masked within the cluster of similarly sized, small subunits. Eucaryal and bacterial homologs of the M. thermoautotrophicum ΔH subunits are listed based on motif conservation and sequence alignments (5, 16, 21, 22). Members of the MTH1324 family (subunit F) have limited similarity to eucaryal Rpb4, a nonessential subunit of RNAPII that in yeast participates in transcription under stress conditions and at temperature extremes (4, 20). All members of the MTH0680.5 family (subunit P) contain four cysteinyl residues arranged in a manner consistent with a C-4 zinc finger motif and homology to Rpb12 (see Fig. 2C). .

FIG. 2

Organization and conservation of the rpoF (A) and rpoP (B) regions in the genomes of M. thermoautotrophicum (MT), A. fulgidus (AF), M. jannaschii (MJ), P. horikoshii (PH), and P. aerophilum (PA) (2, 6, 11, 12, 23). Arrows indicate directions of transcription, shading patterns show homology between genes, and broken lines indicate nonadjacent locations. MTH1323 and MTH0681 and their homologs are predicted to encode rpL21 and rpL37a, respectively. MTH1325 and MTH0680 gene products and their homologs are unknown. Amino acid sequences (C) of MTH0680.5 (MT) and MJ0593.5 (MJ) aligned with their archaeal homologs and the sequences of Rpb12 from Saccharomyces cerevisiae (SC), Schizosaccharomyces pombe (SP), and Homo sapiens (HS) (21, 22). Identical and similar (indicated by asterisks) amino acid residues are identified in the conserved sequence. Four cysteinyl residues predicted to form a C-4 type of zinc finger are boxed. The numbers of amino acid residues present, but not shown, at the N termini of the eucaryal proteins are indicated by the numbers in parentheses. The PH homolog may have longer N-terminal sequences initiated 28 codons upstream at an in-frame ATG (11).

FIG. 3

(A) Template DNA, obtained by DdeI digestion of plasmid pRT74 (25), and origin of the 193-nt runoff transcript. (B) Autoradiogram of in vitro-synthesized transcripts. A plus sign indicates that a reaction mixture contained the protein listed. Lane S, size standards; nt, nucleotides.

FIG. 4

Sucrose gradient cosedimentation of M. thermoautotrophicum ΔH RNAP and TFB and immunoprecipitation of RNAP by anti-TFB antibodies. Fractions containing TFB and TBP were identified by Western blotting. The inset shows the results of immunoprecipitation experiments with protein A-Sepharose preparations lacking antibodies (−) or coupled to anti-TBP (α-TBP) or anti-TFB (α-TFB). The average values for four separate experiments are shown, with the RNAP activity eluted from each matrix calculated as a percentage of the RNAP activity eluted from the matrix carrying anti-TFB antibodies.