Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12 - PubMed (original) (raw)

Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12

F Werner et al. Nucleic Acids Res. 2000.

Abstract

The archaeal and eukaryotic evolutionary domains diverged from each other approximately 2 billion years ago, but many of the core components of their transcriptional and translational machineries still display a readily recognizable degree of similarity in their primary structures. The F and P subunits present in archaeal RNA polymerases were only recently identified in a purified archaeal RNA polymerase preparation and, on the basis of localized sequence homologies, tentatively identified as archaeal versions of the eukaryotic RPB4 and RPB12 RNA polymerase subunits, respectively. We prepared recombinant versions of the F and P subunits from Methanococcus jannaschii and used them in in vitro and in vivo protein interaction assays to demonstrate that they interact with other archaeal subunits in a manner predicted from their eukaryotic counterparts. The overall structural conservation of the M. jannaschii F subunit, although not readily recognizable on the primary amino acid sequence level, is sufficiently high to allow the formation of an archaeal-human F-RPB7 hybrid complex.

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Figures

Figure 1

Figure 1

Sequence alignment of archaeal F and P subunits with eukaryotic RPB4 and RPB12 proteins, respectively. (A) Alignment of two representative archaeal F subunit sequences with two eukaryotic RPB4 subunits (mj, M.jannaschii; mt, M.thermoautotrophicum; sp, Schizosaccharomyces pombe; hs, Homo sapiens). Note the limited degree of overall homology and the rather disperse location of similar or identical present in all four polypeptide sequences shown. (B) Alignment of two representative archaeal P subunit sequences with two eukaryotic RPB12 subunits (species abbreviations as above). Dark shaded residues are identical and light shaded residues are similar.

Figure 2

Figure 2

Coexpression of GST–hsRPB4 and hsRPB7 subunits in E.coli. (A) Schematic representation of the expression constructs used. (B) Comparison of the protein expression profiles of bacterial cells harboring the constructs illustrated above. The insoluble and soluble fractions, together with the glutathione column eluates (GCE), are shown for the GST–hsRPB4 (lanes 1, 4 and 7), GST–hsRPB7 (lanes 2, 5 and 8) and GST–hsRPB4/hsRPB7 (lanes 3, 6 and 9) expression strains. The GST–hsRPB4 and GST–hsRPB7 fusion proteins are highly insoluble when expressed by themselves and are thus exclusively present in the pellet fractions (lanes 1 and 2). The bicistronic construct expressing GST–hRPB4 and hRPB7 leads, however, to the formation of a heterodimeric complex with increased solubility that can be purified by glutathione affinity chromatography (lane 9).

Figure 3

Figure 3

Coexpression of GST–mjF and mjE subunits in E.coli. (A) Schematic representation of the expression constructs used. (B) Comparison of the protein expression profiles of bacterial cells harboring the constructs illustrated above. The insoluble and soluble fractions, together with GCE, are shown for the GST–mjF (lanes 1, 4 and 7), GST–mjE (lanes 2, 5 and 8) and GST–mjF/mjE (lanes 3, 6 and 9) expression strains. The GST–mjE fusion protein is highly insoluble when expressed on its own and is thus exclusively present in the pellet fractions (lane 2). The bicistronic construct expressing GST–mjF and mjE leads, however, to the formation of a heterodimeric complex with increased solubility that can be purified by glutathione affinity chromatography (lane 9).

Figure 4

Figure 4

Copurification of mjF–mjE. (A). Thrombin-cleavage of the affinity purified GST–mjF/mjE complex releases the GST moiety from mjF, resulting in a mixture of three polypeptides (lane L). GST does not bind to DEAE–Sepharose (lane FT, flowthrough; lane W, wash fractions), whereas mjE and mjF coelute with each other as the ionic strength in the elution buffer is increased. (B) Native size exclusion chromatography of the mjF–mjE complex. Eluates from the high-salt fractions from the DEAE–Sepharose purification step were chromatographed on an S-100HR size exclusion column. mjE and mjF, despite their different sizes, coelute as a symmetrical peak migrating in a position consistent with their combined molecular weights.

Figure 5

Figure 5

Assembly of an archaeal–human hybrid complex. (A) Schematic representation of the expression constructs used. (B) Analysis of the protein expression profiles of bacterial cells harboring the constructs illustrated above. The insoluble and soluble fractions, together with GCE, are shown for the GST–mjF/mjE (lanes 1, 3 and 5) and GST–mjF/hsRPB7 (lanes 2, 4 and 6) expression strains. The cross-species bicistronic construct expressing GSTmjF and hsRPB7 expresses an archaeal–human hybrid complex (lane 6). Partial GST–mjF products present in this lane are indicated by an asterisk.

Figure 6

Figure 6

The yeast two-hybrid system demonstrates a specific in vivo interaction between mjP and mjD. β-Galactosidase assay of yeast strains cotransformed with the indicated GAL4 activation (AAD) and GAL4 DNA-binding (DBD) domain fusion expression plasmids. The β-galactosidase activity produced in the cells containing the various plasmid combinations shown are as a percentage relative to the p53/large T positive control.

Figure 7

Figure 7

Formation of a heterotetrameric mjD–mjL–mjN–mjP complex. Tricine SDS–PAGE gel of size exclusion chromatography fractions of the mjD–mjL–mjN–mjP complex run on an S-100HR size exclusion chromatography column (Amersham-Pharmacia Biotech).

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