1. Distinct Roles for CTD Ser-2 and Ser-5 Phosphorylation in the Recruitment and Allosteric Activation of Mammalian mRNA Capping Enzyme 
C.Kiong Ho, Stewart Shuman 
Molecular Cell 
Volume 3, Issue 3, Pages (March 1999)
DOI: /S (00)

2. Figure 1
Binding of Mammalian Guanylyltransferase to a Defined Synthetic CTD Phosphopeptide
(A) The structure of the CTD peptide with “n” number of tandem heptad repeats containing phosphoserine at position 5 is shown. An N-terminal biotin anchors the peptide to a streptavidin-coated magnetic bead.
(B) CTD affinity chromatography. Mouse guanylyltransferase was incubated with either streptavidin beads alone (control) or beads with immobilized CTD-PO4 with two (n = 2), four (n = 4), or six (n = 6) tandem heptad repeats. CTD affinity chromatography was performed as described in Experimental Procedures. Aliquots of the input guanylyltransferase protein fraction (I) (equivalent to 40% of total material loaded), free-unbound fraction (F) (40% of supernatant), and the bead-bound fraction (B) (40% of SDS eluate) were analyzed by SDS-PAGE. A Coomassie blue stained gel is shown. The positions of the guanylyltransferase (GTase) and streptavidin polypeptides are denoted by arrows on the right. The positions and sizes (in kDa) of marker polypeptides are indicated on the left.
(C) Elution of guanylyltransferase from the CTD phosphopeptide by salt. Mce1(211–597)p was incubated for 30 min with streptavidin beads containing immobilized CTD phosphopeptide (n = 6). After centrifugation and removal of the supernatant, the beads were eluted serially by resuspension and centrifugation in 50 μl of binding buffer containing 50, 100, 250, and 500 mM NaCl. Aliquots (1 μl) of each eluate fraction was assayed for guanylyltransferase activity. The signal intensity of the enzyme-[32P]GMP complex (PSL, photo stimulatable luminescence) is plotted for each step eluate fraction (mM NaCl).
Molecular Cell 1999 3, DOI: ( /S (00) )

3. Figure 2
Phosphorylation of Ser-2 or Ser-5 Confers Guanylyltransferase Binding to the CTD
Mouse guanylyltransferase Mce1(211–597)p (A) and mouse RNA triphosphatase Mce1(1–210)p (B) were subjected to affinity chromatography in 50 mM NaCl buffer using beads with immobilized CTD peptides consisting of four heptad repeats: either unphosphorylated (no PO4), phosphorylated at Ser-2 (Ser-2-PO4), or Ser-5 (Ser-5-PO4). Aliquots comprising 40% of the input enzyme (I), 40% of the unbound supernatant (F), and 40% of the and bead-bound SDS eluate fraction (B) were analyzed by SDS-PAGE. Coomassie blue stained gels are shown. The guanylyltransferase (GTase), triphosphatase (TPase), and streptavidin proteins are indicated by arrows.
Molecular Cell 1999 3, DOI: ( /S (00) )

4. Figure 3
CTD Phosphorylation on Ser-5 Stimulates the Guanylyltransferase Activity of Mammalian Capping Enzyme
(A) Mce1(211–597)p (20 pmol) was incubated for 30 min on ice with 400 pmol of the indicated CTD peptide (containing the specified number of heptads and either no phosphate [−] or phosphoserine at position 2 or 5 as indicated) in 10 μl of binding buffer containing 25 mM NaCl. A control reaction was incubated in parallel without CTD peptide. An aliquot (1 μl) of each mixture was then assayed for enzyme–GMP complex formation. The signal intensities were quantitated with a phosphorimager and then normalized to that of the no CTD control reaction (defined as 1.0). The data shown are averages of two independent experiments.
(B) Concentration dependence of guanylyltransferase activation by CTD Ser-5 phosphopeptides. Mce1(211–597)p (20 pmol) was preincubated with 0, 25, 50, 100, 200, or 400 pmol of the indicated CTD Ser-5-PO4 peptide (n = 2, 4, or 6) in 10 μl of binding buffer containing 25 mM NaCl. An aliquot (1 μl) of each mixture was then assayed for enzyme–GMP complex formation. The fold stimulation is plotted as a function of the final concentration of peptide in the guanylyltransferase reaction, which contained 0.1 μM Mce1(211–597)p.
(C) Mce1(211–597)p (20 pmol) was incubated for 10 min on ice with 0, 2, or 4 nmol of the CTD Ser-2-PO4 peptide (n = 4) in 8 μl of binding buffer containing 25 mM NaCl. Aliquots (2 μl) of buffer containing 0 or 200 pmol of the CTD Ser-5-PO4 peptide (n = 4) were added to the samples, and incubation was continued for 20 min on ice. A control reaction was incubated in parallel without CTD peptides. An aliquot (1 μl) of each sample was then assayed for enzyme–GMP complex formation. The signal intensities were normalized to that of the no CTD control reaction. The final concentrations of the CTD peptides in the guanylyltransferase reaction are indicated below the graph. The data shown are averages of two independent experiments.
(D) GTP titration. Mce1(211–597)p (20 pmol) was preincubated with 400 pmol of the n = 6 CTD Ser-5-PO4 peptide in 10 μl of binding buffer containing 25 mM NaCl. A control sample contained 20 pmol of Mce1(211–597)p alone (−CTD peptide). An aliquot (1 μl) of each sample was assayed for enzyme–GMP complex formation in reaction mixtures containing 50 mM Tris HCl (pH 8.0), 5 mM MgCl2, 5 mM DTT, 0.1 μM Mce1(211–597)p, 0 or 2 μM CTD phosphopeptides, and [α32P]GTP as specified. Enzyme–GMP complex formation (pmol) is plotted as a function of GTP concentration.
Molecular Cell 1999 3, DOI: ( /S (00) )

5. Figure 4
CTD-Ser-5 Phosphopeptides Stimulate the Full-Length Mammalian Capping Enzyme Mce1p
Recombinant full-length mouse capping enzyme Mce1p (6 pmol), purified as described by Ho et al. 1998a, was incubated for 30 min on ice with 120 pmol of the indicated CTD peptide in 6 μl of binding buffer containing 25 mM NaCl. A control reaction was incubated in parallel without CTD peptide. An aliquot (2 μl) of each mixture was then assayed for enzyme–GMP complex formation. The signal intensities were normalized to that of the no CTD control reaction (defined as 1.0). The data shown are averages of two independent experiments.
Molecular Cell 1999 3, DOI: ( /S (00) )