We have examined the properties of two Drosophila RNA polymerase II mutants, C4 and S1, during elongation, pyrophosphorolysis, and DmS-II-stimulated transcript cleavage. The C4 and S1 mutants contain a single amino acid substitution in the largest and second largest subunits, respectively. Compared with wild type, C4 had a lower elongation rate and was less efficient at reading through intrinsic elongation blocks. S1 had a higher elongation rate than wild type and was more efficient at reading through the same blocks. During elongation, C4 and wild type responded similarly to DmS-II and NH4+ whereas the S1 mutant was less responsive to both. Differences between the two mutants also appeared during DmS-II-mediated transcript cleavage and pyrophosphorolysis. During extended pyrophosphorolysis, S1 polymerase was fastest and C4 polymerase was slowest at generating the final pattern of shortened transcripts. S1 and wild type were equal in the rate of extended DmS-II mediated transcript cleavage, and C4 was slower. Our results suggest that the S1 mutation increases the time spent by the polymerase in elongation competent mode and that the C4 mutation may affect the movement of the polymerase.

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thrl24, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Grlsser, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13 -221. The peptide, A-D-S-Q-H-S-T-P-P, which corresponds to the amino acid sequence 11 8 -125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the -4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the -2 and -3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent K, values around 2 mM and V,,, values up to 500 pmol . min-. ml-'. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first-order rate constant over 20-fold higher than peptides with acidic amino acid residues at the N-terminus only; the apparent K, value was 0.65 mM with a V,,, of 2900 pmol . min-' . ml-'. The effects of modifying Serl20 to phosphoserine were examined by addition of a recognition sequence for the CAMP-dependent protein kinase prior to Serl20. Prior phosphorylation of the peptide at Serl20 lowered the apparent K, to 0.061 mM and increased the V,,, to 360 pmol . min-' . ml-', a 50-fold decrease in K , for casein kinase I and a 6-fold increase in V,,, as compared to the non-phosphorylated peptide. This indicates that Serl20, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.Casein kinase I is a second-messenger-independent protein kinase which phosphorylates serine and threonine residues in an acidic environment [l, 21. Casein kinase I is a monomer and is present in the nucleus, cytoplasm and membrane of all eukaryotic cells examined. The enzyme from reticulocytes has a molecular mass ranging over 34-37 kDa, depending on the Early studies examining phosphorylation of casein variants by casein kinase I, illustrated the effectiveness of glutamate residues at the -2 position preceding the phosphorylatable serine for recognition by casein kinase I [5]. However, the sites phosphorylated on SV40 large T antigen and glycogen synthase by casein kinase I lack an acidic residue at the -2 position. For glycogen synthase, the critical residue has been shown to be serine phosphate at the -3 position relative to the phosphorylatable serine [6]. Phosphorylation In this study, a number of synthetic peptides related to the region containing Serl23 from SV40 large T antigen were examined to further define the determinants recognized by casein kinase I. The kinetics of phosphorylation were studied to determine the relative effectiveness of acidic residues at the -2 an...

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