Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation
Figure 4
Cka1 disrupts the ability of Npl3 to effectively compete for binding to an Rna15-preferred sequence.
(A) Recombinant Npl3, Rna15 and Cka1 were incubated with a radiolabeled RNA oligo (N4), UV cross-linked, and resolved in denaturing 10% SDS-PAGE gels. This RNA oligo consists of an A-rich repeat (described in Materials and Methods), which is the preferred binding site for Rna15 and is commonly found upstream of polyA sites [7]. Representative UV-cross-linking experiments are shown where increasing Cka1 is added to reactions containing Npl3 with Rna15. The graph below each gel shows quantification for the average of three experiments (Npl3, white bars; Rna15, black bars). Binding levels were calculated as fractions relative to a reaction containing the highest concentration of the individual RNA-bound protein (lane 1 for Npl3 and lane 2 for Rna15). Control UV-crosslinking experiment where Cka1 has been added with or without ATP is also shown. Values represent total PhosphorImager units (PIU). (B) Recombinant Npl3, Rna15 and Cka1 were incubated with a radiolabeled U/G/C-rich RNA oligo (N2), UV-cross-linked, and resolved in denaturing 10% SDS-PAGE gels and quantified as described for (A). (C) Similar reactions were performed as described for Figure 1 for 0 and 30 minutes without (lane 1 and 5) or with 78 nM Npl3 (lanes 2–3 for 0 min time-point; and 6–7 for the 30 min time-point). 200 nM Rna15 was added to the transcription reactions in the presence or absence of Npl3 (lanes 3–4 for 0 min time-point; and 7–8 for the 30 min time-point). The oligo(dC)-template is represented next to the gel and the positions of the pause sites at stretches of Ts are indicated. Quantification of the transcription reactions was performed as shown for Figure 2C.