RNA polymerase II, the polymerase responsible for transcribing protein coding genes in eukaryotes, possesses an ability to discriminate between correct (complementary to the DNA template) and incorrect substrates (selectivity), and as well as remove incorrect substrates that have been erroneously incorporated into the nascent RNA transcript (proofreading). Although these features of pol II are not as robust as those observed for DNA polymerases, the accurate utilization of genetic information is of obvious importance to the cell. The role of the small RNA polymerase II subunit Rpb9 in transcriptional proofreading was assessed in vitro. Transcription elongation complexes in which the 3'-end of the RNA is not complementary to the DNA template have a dramatically reduced rate of elongation, which provides a fidelity checkpoint at which the error can be removed. The efficiency of such proofreading depends on competing rates of error propagation (extending the RNA chain without removing the error) and error excision, a process that is facilitated by TFIIS. In the absence of Rpb9, the rate of error propagation is increased by 2- to 3-fold in numerous sequence contexts, compromising the efficiency of proofreading. In addition, the rate and extent of TFIIS-mediated error excision is also significantly compromised in the absence of Rpb9. In at least some sequence contexts, Rpb9 appears to enhance TFIIS-mediated error excision by facilitating efficient formation of a conformation necessary for RNA cleavage. If a transcription error is propagated by addition of a nucleotide to the mismatched 3'-end, the rate of further elongation increases but remains much slower than that of a complex with a fully base-paired RNA, which provides a second potential fidelity checkpoint. The absence of Rpb9 also affects both error propagation and TFIIS-mediated error excision at this potential fidelity checkpoint in a manner that compromises transcriptional fidelity. The trigger loop, a mobile structural element of the largest subunit of RNA polymerase II is important for maintaining fidelity. The pol II specific toxin α-amanitin targets the trigger loop, and was used to distinguish trigger loop -independent and -dependent Rpb9 functions, in vitro. Rpb9 decreases the correct nt extension rate when trigger loop movement is restricted by α-amanitin. This occurs in the context of a RNA with a matched or mismatched 3’-end, which indicates that Rpb9’s contribution to correct nt extension occurs in a manner independent of the trigger loop. In addition, the effect on mismatch extension indicates that the trigger loop is not required for Rpb9 to facilitate recognition of proofreading ‘checkpoints’ after mismatches occur. Rpb9 also decreases the rate of misincorporation, but this effect is dependent on the trigger loop. Rpb9’s role in selectivity was tested by utilizing several assays to estimate nt discrimination. Rpb9 does not have a significant effect on nt discrimination for the sequence contexts tested, which suggests the role Rpb9 plays in fidelity is in large part due to its proofreading capabilities. Lastly, the charged residues of Rpb9’s C-terminal “loop” region, proposed in the prevailing model to be important for trigger loop interaction, are dispensable for Rpb9 function in vivo and in vitro.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (Pol II) is the primary actor in the transcription of mRNA from genes. Pol II is a complex composed of twelve protein subunits. This study focused on the changes in the interactome of Rbp3 in S. cerevisiae when the Pol II subunit Rpb9 is removed. Rpb3 is one of the core subunits of Pol II, and any significant changes to the Rpb3 incteractome due to the loss of Rpb9 can be used to infer new information about Rpb9’s role in the Pol II complex. Rpb3 was pulled down using FLAG purification from both wild type and rpb9Δ S. cerevisiae cultures. Rpb3 and the proteins complexed with it were then analyzed using multi-dimensional protein identification technology (MudPIT), a form of liquid chromatography-mass spectrometry (LC-MS). This data was searched using the SEQUEST database search algorithm, and the results were further analyzed for likelihood of interaction using Significance Analysis of INTeractome (SAINT), as well as for post-translational phosphorylation. Deletion of rpb9 did not present any changes in Pol II phosphorylation however it did cause several changes in the interaction network. The rpb9Δ strain showed new interactions with Rtr1, Sen1, Vtc4, Pyc1, Tgl4, Sec61, Tfb2, Hfd1, Erv25, Rib4, Sla1, Ubp15, Bbc1, and Hxk1. The most prominent of these hits are Rtr1, an Rpb1 C-terminal domain phosphatase linked to transcription termination, and Sen1, an RNA/DNA nuclease that terminates transcription. In addition, this mutant showed no interaction with Mtd1, an interaction that is present in the wild type. In all cases, these hits should be considered fuel for future research, rather than conclusive evidence of novel interactions.