Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

DNA contains the instructions for making proteins, which build and maintain our cells. So that the information encoded in DNA can be used, a molecular machine called RNA polymerase II makes copies of specific genes. These copies, in the form of a molecule called RNA, convey the instructions for making proteins to the rest of the cell. To ensure that RNA polymerase II copies the correct genes at the correct time, a group of regulatory proteins are needed to control its activity. Many of these proteins interact with RNA polymerase II at a region known as the C-terminal domain, or CTD for short. For example, before RNA polymerase can make a full copy of a gene, a small molecule called a phosphate group must first be added to CTD at specific units known as Ser2. The regulatory protein P-TEFb was thought to be responsible for phosphorylating Ser2. However, it was previously not known how P-TEFb added this phosphate group, and why it did not also add phosphate groups to other positions in the CTD domain that are structurally similar to Ser2. To investigate this, Mayfield, Irani et al. mixed the CTD domain with different regulatory proteins, and used various biochemical approaches to examine which specific positions of the domain had phosphate groups attached. These experiments revealed a previously unknown aspect of P-TEFb activity: its specificity for Ser2 increased dramatically if a different regulatory protein first added a phosphate group to a nearby location in CTD. This additional phosphate group directed P-TEFb to then add its phosphate specifically at Ser2. To confirm the activity of this mechanism in living human cells, Mayfield, Irani et al. used a drug that prevented the first phosphate from being added. In the drug treated cells, RNA polymerase II was found more frequently ‘stalled’ at positions on the DNA just before a gene starts. This suggests that living cells needs this two-phosphate code system in order for RNA polymerase II to progress and make copies of specific genes. These results are a step forward in understanding the complex control mechanisms cells use to make proteins from their DNA. Moreover, the model presented here – one phosphate addition priming a second specific phosphate addition – provides a template that may underlie similar regulatory processes.