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Molecular and Cellular Biology, September 2000, p. 7013-7023, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identifying a Core RNA Polymerase Surface Critical for Interactions with a Sigma-Like Specificity Factor

Paul F. Cliften,1,dagger Sei-Heon Jang,2 and Judith A. Jaehning1,*

Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262,1 and Department of Molecular Biology, Taegu University, Taegu, Korea2

Received 14 April 2000/Returned for modification 22 May 2000/Accepted 7 June 2000

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 of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In 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 of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, B121, UCHSC, 4200 E. Ninth Ave., Denver, CO 80262. Phone: (303) 315-3004. Fax: (303) 315-3326. E-mail: Judith.Jaehning{at}UCHSC.edu.

dagger Present address: Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110.

Molecular and Cellular Biology, September 2000, p. 7013-7023, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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