The beta ' subunit of Escherichia coli RNA polymerase is not required for interaction with initiating nucleotide but is necessary for interaction with rifampicin.

Using a modification of a highly selective affinity labeling protocol, we demonstrated that the alpha(2)beta subassembly of Escherichia coli RNA polymerase efficiently and specifically interacts with the initiating purine nucleotide. Isolated beta is also active in this reaction. In contrast, neither beta nor alpha(2)beta is able to interact with a chimeric molecule composed of rifampicin attached to an initiation substrate. Based on these results, we conclude that the RNA polymerase initiation site, specific for purine nucleotides, which ultimately become the 5'-end of the transcript, is essentially complete in the absence of the largest subunit, beta'. However, the rifampicin binding center is formed only in the alpha(2)betabeta' core enzyme. We interpret our results in light of the high resolution structure of core RNA polymerase from Thermus aquaticus.

Inter- and intrasubunit interactions during the formation of RNA polymerase assembly intermediate.

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) alpha and beta subunits during the formation of alpha(2)beta, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing beta conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with alpha. The alpha-interacting domain is held together by protein-protein interactions between beta segments F and I. Residues in catalytically important beta segments H and I directly participate in alpha binding; substitutions of strictly conserved segment H Asp(1084) and segment I Gly(1215) abolish alpha(2)beta formation in vitro and are lethal in vivo. The importance of these beta amino acids in alpha 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 beta Asp(1084) in A135, the beta-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest alpha-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli alpha-beta 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.

A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly.

All multisubunit DNA-dependent RNA polymerases (RNAP) are zinc metalloenzymes, and at least two zinc atoms are present per enzyme molecule. RNAP residues involved in zinc binding and the functional role of zinc ions in the transcription mechanism or RNAP structure are unknown. Here, we locate four cysteine residues in the Escherichia coli RNAP largest subunit, beta', that coordinate one of the two zinc ions tightly associated with the enzyme. In the absence of zinc, or when zinc binding is prevented by mutation, the in vitro-assembled RNAP retains the proper subunit stoichiometry but is not functional. We demonstrate that zinc acts as a molecular chaperone, converting denatured beta' into a compact conformation that productively associates with other RNAP subunits. The beta' residues coordinating zinc are conserved throughout eubacteria and chloroplasts, but are absent from homologs from eukaryotes and archaea. Thus, the involvement of zinc in the RNAP assembly may be a unique feature of eubacterial-type enzymes.

Localization of Escherichia coli rpoC mutations that affect RNA polymerase assembly and activity at high temperature.

We localized five rpoC (beta') mutations affecting Escherichia coli RNA polymerase assembly. The Ts4, XH56, and R120 mutations changed beta' residues conserved throughout eubacteria; the JE10092 mutation occurred in the hypervariable region; rpoC1 (TsX) changed a universally conserved residue and corresponds to yeast rpb1-1. Thus, distinct, predominantly conserved beta' residues participate in interactions holding RNA polymerase together.