Large Terminase Protein Research Articles

Defining the ATPase Center of Bacteriophage T4 DNA Packaging Machine: Requirement for a Catalytic Glutamate Residue in the Large Terminase Protein gp17

Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT 161–167) and Walker B (MIYID 251–255) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine.

The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4

Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured. Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown. Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4. This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs. The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome. The precise mutation giving rise to a particular phenotype was determined by DNA sequencing. The data showed that the N-terminal ATP binding site I (SRQLGKT161–167), but not the ATP binding site II (TAAVEGKS299–306), is critical for gp17 function. Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate. Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity. The purified K166G mutant showed a loss of gp17-ATPase activity. The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging.

Expression of the bacteriophage T4 DNA terminase genes 16 and 17 yields multiple proteins

The products of the bacteriophage T4 terminase genes 16 and 17 are known to mediate cutting and packaging of concatemeric vegetative DNA. We show here that the larger of these genes, 17, yields multiple protein species. The complex expression of the T4 terminase genes includes overlapping transcripts, probably initiated from multiple promoters, RNA processing at certain preferred sites and translation initiation from multiple ribosome binding sites (RBS). Translation initiation from these RBS may be modulated by inverted repeat (IR) sequences whose folding can be predicted to differ in different RNA species. In T4 infected bacteria, genes 16 and 17 are probably co-transcribed from several near-consensus late promoters upstream from gene 16, and processed at multiple sites. Additional 5 ′ ends of late transcripts are located downstream from a near-consensus late promoter inside gene 17 and further downstream, unrelated to any known promoter consensus sequence. The gene 17 transcripts that are initiated or cleaved internally contain RBS for shorter open reading frames (ORFs) in the same frame as full-length gene product (gp) 17 of 70 kDa. The truncated proteins, a 59-kDa gp17 ′ and a 45-kDa gpl7 ″, are synthesized from cloned gene 17 segments in which the first gene 17 RBS is deleted. Expression of gene 17 is different in BL21(DE3) or W3110[pACT7] host bacteria. The gp17 ′ and gp17 ″ proteins are predicted to contain one or more of the ATPase motifs that are common among large subunits of other phage terminases. They lack a predicted single stranded (ss) DNA binding motif that is unique the large terminase proteins in T4 gpl7, and that has been implicated in recognizing ssDNA regions in replicating and recombining T4 DNA destined to be packaged. We hypothesize that a truncated gene 17 ′ is an evolutionary precursor of the full-size T4 gene 17. Its function may have been maintained to allow processive packaging from double stranded (ds) DNA ends.