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J. Biol. Chem., Vol. 275, Issue 40, 31183-31190, October 6, 2000

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Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate^*

Tatyana Naryshkina, Dragana Rogulja^§¶, Larisa Golub^§, and Konstantin Severinov^**

From the Waksman Institute for Microbiology and the  Department of Genetics, Rutgers, State University of New Jersey, Piscataway, New Jersey 08854

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP)  and  subunits during the formation of ₂, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing  conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with . The -interacting domain is held together by protein-protein interactions between  segments F and I. Residues in catalytically important  segments H and I directly participate in  binding; substitutions of strictly conserved segment H Asp¹⁰⁸⁴ and segment I Gly¹²¹⁵ abolish ₂ formation in vitro and are lethal in vivo. The importance of these  amino acids in  binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of  Asp¹⁰⁸⁴ in A135, the -like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest -like subunit. However, the A135-AC40 interaction is weak compared with the E. coli - interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.

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