Pcf11p contains a CTD-binding domain and interacts directly with the
CTD. (A) Schematic representation of the domain
organization of Pcf11p. The CTD-binding domain is indicated as CTD-BD.
(B) Alignment of conserved CTD-binding domains. The
N-terminal CTD-binding domain of S. cerevisiae Pcf11p is
compared with domains of homologous proteins from S.
pombe (SPAC4G9.04C gene product; GenBank accession no. Z69727),
Neurospora crassa (B1D1.390 gene product; no. CAB91288),
C. elegans (CELR144.2 gene product; no. U23515),
Drosophila melanogaster (CG10228 gene product; no.
AAF58192), Homo sapiens (Pcf11 homolog; no. AF046935),
Arabidopsis thaliana (T4B21.1 gene product; no.
AAD03447), S. cerevisiae Nrd1p (no. CAA96158),
Rattus norvegicus rA4 (no. U49058), and R.
norvegicus SCAF8 (no. U49055). Bold residues indicated with
asterisks are identical among at least five of the sequences. In
addition to these residues, there are many similarities that are not
highlighted. The gaps introduced to maximize the homology are
represented by dashed lines. (C) Pcf11p interacts
directly with the CTD, whereas Rna15p does not. Recombinant His-tagged
Pcf11p and Rna15p were chromatographed on GST, GST-CTD, and
phosphorylated GST-CTD resins. The bound fraction was eluted with SDS
sample buffer and detected by Western blot with antihistidine
monoclonal antibodies. Load (100% of input), pellet (100%), and
supernatant (10%) fractions are shown. Lanes 1–3, Rna15p
chromatography; lanes 4–6, Pcf11p chromatography. The lower bands
detectable in lanes 5 and 6 are degradation products of full-length
Pcf11p.