Proteomics mapping of Yeast RNA Polymerase II Post-translational modifications and investigating their biological significance

摘要

RNA polymerase II (RNAP II) has multiple roles in transcription and plays a role in gene expression by controlling selective recruitment of chromatin, and also RNA processing factors. Despite a wide interest in RNAP II, the only region of RNAP II in which post-translational modifications have been extensively studied is the unstructured C terminal domain. In order to ask whether other regions of the enzyme were modified, we analyzed yeast RNAP II peptides modifications by MudPIT (Multi-dimensional protein identification technology). We identified several lysine modified residues in RNAP II and highly seen modification is acetylation which is almost 50percent of total modifications identified. Yeast strains encoding either RNAP II subunit Rpb3 or Rpb11 with a C terminal TAP-tag were used to affinity purify RNAP II containing protein complexes. These were digested by three proteases (trypsin, chymotrypsin, and Glu-C) and the resulting peptide mixtures were analyzed with an LTQ Orbitrap Velos using collision induced dissociation. We did three technical replicates for each biological replicate sample to minimize the experimental error and the total number of samples analyzed is 27. The spectra were processed with Raw Distiller and subsequent data analysis was carried out using SEQUEST. The protein lists were established based on conservative filtering criteria (DeltCn of 0.08, minimum Xcorr of 1.8 for +1, 2.5 for +2, and 3.5 for +3 charged peptides, and have a minimum peptide length of 7 amino acids). The peptide mass tolerance was set to 7 ppm. All strains used for the subsequent screening experiments were made as follows. Firstly we constructed a yeast strain with the gene for the relevant RNAP II subunit deleted in the genome, and containing a wild type copy of the gene on a plasmid. Secondly, we transformed this strain with a second, mutated copy of the gene encoding the subunit. We were then able to remove the wild type copy of the gene to ask whether the strain was viable in the presence of only the mutated gene. The plasmids were prepared by transferring wild type and mutant genes from the pENTR vector to gateway destination vectors using an LR clonase reaction. Preliminary data We found many novel post-translational modifications in RNAP II by mapping the 12 subunits of the complex using three different proteases. The data shown here is for the modifications which are seen at least in two biological replicates. We identified total 211 modifications in 12 subunits of RNA polymerase II. Mostly seen modification is acetylation and the highly modified amino acid is lysine. Most of the modifications were seen in Rpb1 and Rpb2 subunits which are 68percent of total number of modifications. New phosphorylation (11) events were also seen which were not reported before. Highly modified lysine residues within the Rpb1, Rpb2, Rpb3, Rpb4, Rpb7, and Rpb11 subunits were selected and systematically mutated them to arginine. The biological screening experiments are under progress. Novel Aspect We have systematically defined the posttranslational modifications of RNA Polymerase II and determining the biological significance of these modifications.