RNA polymerase II elongation study along yeast cell cycle.

Increasing evidence supports that the activity of RNA polymerase II (RNA pol II) during transcription elongation can be regulated to control transcription rates. Using genomic run-on (GRO) and RNA pol II chromatin immunoprecipitation (RPCC), we have respectively measured active and total RNA pol II present in the body of genes at 3 different stages of the mitotic cell cycle of Saccharomyces cerevisiae: G1, S and G2/M. Comparison of active and total RNA pol II values at these three points defined several gene clusters that reflect different patterns of transcription elongation control across the cell cycle. Previously defined cycling genes were overrepresented in some of these clusters, which showed cyclic average mRNA expression patterns. One of the clusters with most divergent GRO and RPCC patterns was significantly enriched in genes functionally related to ribosome biogenesis (RiBi). We confirmed that RiBi mRNA expression upregulates after START. Finally, we analysed the contribution of mRNA stability to each cluster and found that concerted control of RNA pol II activity and mRNA decay is needed to fully understand alternative strategies of gene expression across the cell cycle. After alpfa-factor synchronization cells were taken for GRO and RPCC analysis at three times: 0 (start), 30 min and 60 min after alpha-factor release. GRO samples were normalized by genomic DNA hybridization of the same macroarray (Filter F1-F7). For RPCC analysis cells were used for chromatin immunoprecitation of RNA pol II using an specific antobody. RPCC samples were obtained by normalization of the IP samples by the WCE samples hybridized in the same macroarray.