Open Complex Contacts: An Analysis of the T5 N25 Discriminator Region Interaction with E. coli RNA Polymerase
E. coli RNA polymerase (RNAP) is known to contact a promoter via the UP element and its -35 and -10 hexameric sequences during the first stage of transcription to transition into a catalytically competent open complex. In this binding-activation process, strong promoter-RNAP interaction provides stability to the open complex, resulting in a high initiation rate. Bacteriophage T5 N25 promoter is such an example where strong contacts at the open complex stage pose a high barrier for escape. As a result, RNAP undergoes repeated abortive cycling releasing short transcripts, prior to ultimately leaving the promoter and transitions into the elongation phase. Recent research by Haugen et al. (2006, 2008) revealed that the discriminator (DIS) region makes an additional direct contact with RNAP through the nontemplate strand G residue positioned two nucleotides downstream of the -10 box, to further stabilize an open complex. Through photocrosslinking analysis of this G residue within the rrnB P1 C-7G and λ PR promoters, their study found that contacts were made to the σ1.2 region of E. coli RNA polymerase. Because the N25 promoter is well know for its open complex stability, we wished to investigate whether this interaction contributes to its stabilization. Photocrosslinking and Western blot analysis completed by J. Wiwczar (2008) revealed that the N25 promoter DIS region contacts the β subunit of RNAP. To investigate the difference in the above results, research was initiated by the construction of N25 promoters that differ in the three nucleotides immediately downstream of the -10 box: AGA in CW1 (wildtype N25), GGA in CW2 (similar to GGT in λ PR) and GGG in CW3 (matching GGG in C-7G rrnB P1). The goal was to determine whether contact to RNAP is a function of DIS region sequence composition. The constructed promoters were thereby subjected to UV-induced crosslinking to RNAP via their -5 S6G residue. Preliminary results show that all three promoters transcribe very similarly, indicating that the stability of the open complex is minimally affected by the DIS mutations. In crosslinking, all three promoters were found to contact the β subunit. To further investigate which amino acids of the β subunit of RNA polymerase are involved in making this contact, we strive to submit the complex for mass spectrometry analysis. To achieve this, the protein-nucleic acid conjugate must be shortened by using restriction enzymes and proteases. We have found that trypsin and Apo I digests together give a reasonably sized complex to be analyzed in the future. In summary, our results indicate that the -5 G nontemplate base within the DIS region of the N25 promoter was contacted by the β subunit independent of the sequence of nucleotides bordering the G residue. To map which amino acids within β are directly involved in contact, a shortened crosslinked complex can be applied for mass spectrometry analysis.