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.
Naryshkina
T
T
Waksman Institute for Microbiology and the Department of Genetics, Rutgers, State University of New Jersey, Piscataway, New Jersey 08854, USA.
Rogulja
D
D
Golub
L
L
Severinov
K
K
D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D009532:Nickel; D012321:DNA-Directed RNA Polymerases