STRUCTURAL AND FUNCTIONAL DOMAINS OF THE LARGE SUBUNIT OF THE BACTERIOPHAGE-T3 DNA PACKAGING ENZYME - IMPORTANCE OF THE C-TERMINAL REGION IN PROHEAD BINDING

During head assembly of phage T3, DNA is packaged into a preformed pro tein shell, called the prohead, with the aid of non-capsid packaging p roteins, the products of genes 18 and 19 (gp18 and gp19). We have deve loped a defined system, composed of purified gp18, gp19 and proheads f or in vitro packaging of T3 DNA. Our previous results using the define d in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectivel y These complexes associate to form a direct precursor complexes for D NA translocation into the head. The formation of the precursor complex es requires ATP as an allosteric effector. Subsequent DNA translocatio n is driven by ATP hydrolysis. gp19 is an ATP binding protein that pla ys multiple roles in DNA packaging through interaction with ATP. gp19 changes its conformation by binding to ATP, as judged from the analysi s of limited proteolysis. Sites cleaved by limited proteolysis were de termined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP. C-Terminal fragments generated by trypsin digestion bound the prohead and inhibit ed DNA packaging by intact gp19 in a competitive manner. On the other hand, N-terminal fragments did not bind the prohead nor did they inhib it DNA packaging. These results define a prohead binding domain at the C terminus of gp19. To identify the prohead binding domain more preci sely, deletion mutants lacking the last 10 and 15 amino acids (gp19-De lta C10 and gp19-Delta C15, respectively) of the extreme C terminus of gp19 were constructed. Limited tryptic digestion patterns of these mu tant proteins in the presence or absence of ATP were basically the sam e as those of gp19-wt, indicating that the conformation and its ATP re sponse were not changed by these deletions. gp19-Delta C15 lacked proh ead binding activity and, therefore, DNA packaging activity gp19-Delta C10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt. These results indicate that a C-termina l region of residues L571 to D576 of gp19 is crucial for prohead bindi ng and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead.