Mass Spectrometry-based Analysis Of Phosphorylation Pattern Of RNA Polymerase ? CTD By P-TEFb

The RNA polymerase ? is responsible not only for transcription of thousands of protein-coding genes constituting the largest group of distinct individual genes in the eukaryotic genome,but also for genes encoding small noncoding RNAs(snRNA).Furthermore,it is found that the carboxyl-terminal domain(CTD)of RNA polymerase ? is unique to this eukaryotic RNA polymerase.TFIIH and P-TEFb are important general transcription factors involved in the progress of transcription cycle.P-TEFb is not only a kind of general transcription factor,but also one of necessary host transcription factors in HIV-1 gene transcription elongation.Thus,the phosphorylation status of CTD by P-TEFb has attracted great attention for a long time.However,its intrinsic structure with hyperphosphorylation and heptapeptide repeat makes it difficult to be analyzed by using mass spectrometry.In this study,in vitro phosphorylation patterns of full-length CTD by P-TEFb has been studied using liquid chromatography-mass spectrometry(LC-MS).Main contents are as summarized as below:(1)P-TEFb was purify through affinity purification and its kinase activity was verified using synthetic peptide as substrate;(2)9 peptides were detected after tryptic digest in MS analysis,covering 1 to 18 and.228 to 388 amino acids of CTD,meanwhile,remained 19 to 227 amino acids were cleavaged by microwave assisted acid hydrolysis method and 53 peptides were then detected,finally the coverage rate after combination of two parts was up to 92%;(3)phosphorylation status of CTD at different time points was revealed by results of ESI-MS analysis.Firstly,Ser-5 phosphorylation was prefered by P-TEFb followed by Ser-7,further more,if reaction time was long enough,low level phosphorylation of Ser-2 could happened,finally,the sequence after fiftieth heptapeptide repeat have barely been phosphorylated.Generally,in this research,a new method in combination of enzymatic digestion and microwave assisted acid hydrolysis was successfully developed for supplying a new way to study position,frequency and preference of phosphorylation of CTD by P-TEFb directly and accurately.