Biotinylated CTD peptides were coupled to the surface of streptavidin sensor chips, and the specificity of binding of monoclonal antibodies to these peptides was determined using BIACORE technology. A, sequences of biotinylated peptides that were immobilized on streptavidin sensor chips with symbols used in graphs in B–D. B, sensorgrams for the binding of mAb H14 (39 nm) to CTD peptides show that it binds to the 5-phospho and 2 + 5-phospho peptides but not the nonphospho or 2-phospho peptides. C, sensorgrams for the binding of mAb 8WG16 (4 mm) to CTD peptides show that it binds to the nonphospho CTD peptide with the highest steady state RU level. D, sensorgrams for the binding of mAb H5 (1.2 nm) to CTD peptides show that it binds to the 2-phospho, 5-phospho, and 2 + 5-phospho CTD peptides but not to the nonphospho peptide. Sensorgrams shown here are from the lower peptide density chip but are also representative of the specificities observed from the higher peptide density chip.

Serial dilutions of mAb H5 were titrated over the four CTD peptides immobilized on the BIACORE surface. Sensorgrams for binding of mAb H5 to the 2-phospho (A), 5-phospho (B), and 2 + 5-phospho CTD peptides (C) indicate that binding to each of these CTD peptides is proportional to the concentration of antibody. Thick light curves are actual binding sensorgrams, whereas thin dark curves were fit to the binding sensorgrams using BIAEvaluation software. For the 2-phospho and 5-phospho peptides, a simple Langmuir two-component model (A + B = C) was used, whereas the 2 + 5-phospho required the addition of a mass transfer parameter. Inclusion of a mass transfer parameter did not improve the fit for Fig. 2, A or B. Curve fits were used to determine apparent kinetic and equilibrium association and dissociation constants (Table II). Residual plots describe how well the fitted curves match the actual binding sensorgrams (see methods). The top sensorgrams in 2-phospho and 5-phospho plots are a 1:2800 (1.2 nm) dilution of mAb H5 with serial 2-fold dilutions of mAb H5 from top to bottom. Top sensorgram for 2 + 5-phospho plots is a 1:1700 (1.9 nm) dilution of mAb H5 raw ascites fluid with serial 2-fold dilutions of mAb H5 from top bottom. For each peptide, background binding to the nonphospho CTD peptide was subtracted as background. Sensorgrams shown here are generated from titrations on either the low or high peptide density chip but are also representative of the sensorgrams generated on the other chip.

A, 5.0 µg of biotinylated peptides described in Table I were phosphorylated in vitro with CTDK-I and [γ-³²P]ATP. Peptides were pelleted with streptavidin beads and washed extensively in phosphate-buffered saline, and radioactivity incorporation was measured by Cerenkov counting. Radioactivity incorporation (counts/min in streptavidin bead pellet) is plotted against time with S.E. bars for n = 2 time courses. The 2-phospho and 5-phospho CTD peptides were the most heavily phosphorylated by CTDK-I. The nonphospho CTD peptide was also phosphorylated but to a lesser extent compared with the two prephosphorylated peptide substrates. The 2 + 5-phospho CTD peptide was a very poor CTDK-I substrate (data not shown). B, the nonphospho, 5-phospho, and 2 + 5-phospho CTD peptide substrates were phosphorylated by CTDK-I in vitro for 60 min, and HPLC-purified products were analyzed for the presence of phosphoserine, phosphotyrosine, and phosphothreonine after acid hydrolysis. GST-yCTD and purified RNA-PII were also analyzed for phosphoamino acid content. Only phosphoserine was detected in all products analyzed

CTD peptides were phosphorylated by CTDK-I in vitro for 60 min with [γ-³²P]ATP, and products were HPLC-purified. Residues were sequentially removed from the N terminus by Edman degradation (for 17 cycles), and radioactivity (³²P) release was measured for each cycle. Radioactivity release (Cerenkov-counted counts/min) from the peptide is plotted for each corresponding residue. A, Edman degradation of the nonphospho peptide phosphorylated by CTDK-I shows peaks of radioactivity release corresponding to serines 5 and 12 (equivalent to serine 5 of the first and second heptad repeats). B, Edman degradation of the 5-phospho peptide phosphorylated by CTDK-I shows peaks of radioactivity release corresponding to serines 9 and 16 (equivalent to serine 2 of the second and third heptad repeats). C, Edman degradation of the 2-phospho peptide phosphorylated by CTDK-I shows a peak of radioactivity release corresponding to serine 5 of the first heptad repeat.

1.0 µg of each purified GST-CTD fusion protein was phosphorylated by CTDK-I in vitro for 60 min. Reaction mixtures were analyzed by SDS-PAGE on a 4–15% Ready Gel and stained with Coomassie Blue. Phosphorylated products were visualized using a PhosphorImager, and the degree of phosphorylation of each substrate was quantified using ImageQuant software. The extent of phosphorylation of each mutant GST-CTD substrate is normalized to a value of 1.0 for the wild type GST-CTD fusion protein (WT16). Substrates are described in Table I. Lane 1, wild type GST-CTD fusion protein (1.0); lane 2, A214 (YSPTSPS)₁₄ (0.73); lane 3, E²15 (YePTSPS)₁₅ (2.0); lane 4, A515 (YSPTaPS)₁₅ (0.0); lane 5, E518 (YSPTePS)₁₈ (0.46).

After initiation, the TFIIH-associated kinase (Kin28p) phosphorylates the CTD at Ser5. The CTD then becomes a better substrate for phosphorylation by CTDK-I at Ser2. CTDK-I can also efficiently generate doubly phosphorylated CTD from a Ser2-phosphorylated CTD (generated by a CTD phosphatase?) by phosphorylating Ser5. Black circle with white letter, phosphoserine.