THE N-END RULE IN ESCHERICHIA-COLI - CLONING AND ANALYSIS OF THE LEUCYL, PHENYLALANYL-TRANSFER RNA-PROTEIN TRANSFERASE GENE AAT

The N-end rule relates the in vivo half-life of a protein to the ident ity of its N-terminal residue. Distinct versions of the N-end rule ope rate in bacteria, fungi, and mammals. We report the cloning and analys is of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl -tRNA-protein transferase (L/F-transferase), a component of the bacter ial N-end rule pathway. L/F-transferase is required for the degradatio n of N-end rule substrates bearing an N-terminal arginine or lysine. T he aat gene maps to the 19-min region of the E. coli chromosome and en codes a 234-residue protein whose sequence lacks significant similarit ies to sequences in data bases. In vitro, L/F-transferase catalyzes th e posttranslational conjugation of leucine or phenylalanine to the N t ermini of proteins that bear an N-terminal arginine or lysine. However , the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conju gation of leucine but not of phenylalanine to the N terminus of the be ta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approx imately 1 kb from clpA, which encodes a subunit of ATP-dependent prote ase Clp. Although both aat and clpA are required for the degradation o f certain N-end rule substrates, their nearly adjacent genes are conve rgently transcribed. The aat gene lies downstream of an open reading f rame that encodes a homolog of the mammalian multidrug resistance P gl ycoproteins.