Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Rudo Kieft; Yang Zhang; Alexandre P. Marand; Jose Dagoberto Moran; Robert Bridger; Lance Wells; Robert J. Schmitz; Robert Sabatini

Discussion 594 841 carbamidomethylation on cysteine residues and a variable modification of oxidation on methionine 842 residues were enabled to accurately match fragment ions. A fixed value peptide spectral match (PSM) 843 validation node was used to validate each PSM at a maximum delta Cn of 0.05. All PSMs were subjected 844 to further validation with a maximum of 1% false discovery rate utilizing PSMs matched to a reverse 845 (decoy) database while being assigned to protein groups. The L. tarentolae JBP3 gene was amplified from genomic DNA by PCR with primers containing 5' 860 Nde I and 3' BamH I restriction sites. PCR fragments were digested with Nde I and BamH I and 861 subcloned into the pet16b expression vector. Plasmids were transformed into Escherichia coli (BL21 862 DE3), and bacteria were cultured in defined autoinduction media to allow growing cultures to high 863 densities and protein expression without induction, as previously described (121). Briefly, LB media is 864 supplemented with KH 2 PO 4 , Na 2 HPO 4 and 0.05% Glucose, 0.2% Lactose and 0.6% Glycerol. Bacteria 865 were cultured in this media in the presence of 100g/ml ampicillin at 37 O C for 2 h and then shifted to 866 18 O C for 24 h. Cells were lysed and JBP3 purified by metal affinity as previously described for JBP1 (37, . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/753004 doi: bioRxiv preprint