Identifying a core RNA polymerase surface critical for interactions with asigma-like specificity factor

Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions o f the single-subunit yeast mitochondrial RNAP (Rpo41p) important for intera ction with its sigma-like specificity factor (Mtf1p). Previously we found t hat the whole folded structure of both polypeptides as well as specific ami no acids in at least three regions of Mtf1p are required for interaction. I n this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions o f the RNAP which, when modeled on the structure of the closely related phag e T7 RNAP, appear to lie on one surface of the protein. Additional point mu tations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppress ors are found in regions required by T7 RNAP for DNA sequence recognition a nd promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among th e mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specifi city factor, which, like its bacterial counterpart, provides promoter recog nition and DNA melting functions to the holoenzyme.