Protein–Nucleic Acid Interactions 413
with pyrophosphate that is released during the extension reaction. Thus, the RNA polymerase may share
a common two-metal mechanism with the DNA polymerases.
10.8 Machines that Manipulate Duplex DNA
10.8.1 Helicases
Helicases unwind DNA and RNA duplexes, and they participate in nearly every cellular process that involves
nucleic acids. In E. coli, more than a dozen different helicases have been identified, and these carry out a
variety of tasks, ranging from strand separation during DNA replication to more complex processes in
DNA repair and recombination and, in the case of RNA helicases, the degradation of messenger RNA.
Whereas certain repair enzymes might use the energy of DNA binding to flip a single base outside the
duplex, a helicase must not only locally melt the DNA target, but must also move itself along the DNA to pre-
vent re-annealing of the strands. This requires mechanical work, which is provided by the free energy of ATP
binding and hydrolysis. Since helicases must displace proteins from the DNA, they are capable of generat-
ing tremendous force. For instance, helicases can produce sufficient force to displace the protein strepta-
vidin from 5-biotinylated DNA,^47 which binds with a dissociation constant on the order of 10^14 M. There are
several families of DNA helicase in archaea, prokaryotes and eukaryotes, but mechanistically these enzymes
can be divided into two classes according to their direction of translocation. One class moves in the 5–3
direction along single-stranded DNA and the second class moves in the opposite direction, from 3–5.
The first crystal structure of a 3–5helicase was that of PcrA from Bacillis stearothermophilus.^48 The
enzyme consists of several domains, including two that are structurally similar to the ATPase domain of
the recombination protein RecA. Subsequent structures of PcrA helicase in complexes with a DNA sub-
strate have provided details of the unwinding mechanism. There are two separable steps: (1) duplex desta-
bilization, and (2) DNA translocation.^39 Duplex DNA is engaged on the surface of the protein, and one of
the displaced single strands binds to a surface channel. The operation involves allosteric transitions in
which protein sub-domains move relative to each other, so that there are changes in both the surface and
the channel that engage the duplex DNA.
An example of a 5–3helicase is the bacteriophage T7 gene 4 helicase, which is involved in strand sep-
aration during phage DNA replication (Figure 10.19). The protein forms a hexameric ring but, surprisingly,
this ring has twofold rotational symmetry rather than the expected sixfold. ATP hydrolysis around the ring
is associated with a rotation of each subunit about axes perpendicular to the ring. The nucleotide-binding
sites (at the interfaces between the subunits) are therefore non-equivalent, and have differing affinities for the
NTPs. As nucleotide hydrolysis occurs, the states of the three sites may interconvert from NTP-bound to
NDP phosphate binding to empty. This would allow a ‘wave’ of NTP hydrolysis to run around the ring.
This model shows some similarities with the ‘binding-change’ model associated with the F1-subunit of
the mitochondrial ATPase, although the asymmetries arise in different manners (Figure 10.19b).
10.8.2 DNA Pumps
The duplex DNA of bacteriophages is packaged within protein-rich capsid shells at densities approaching
those of crystals. Electrostatic repulsion of the compacted nucleic acid generates pressure, and this force is
used to discharge the DNA into the host cell during the infection process. But the packaging of nucleic acid to
such an extent in the first place requires tremendous work which is provided by motors that are fuelled by ATP
binding and hydrolysis. The force generated by DNA pumps of bacteriophages during the initial stages of
translocation is estimated to be in the order of 100 pico-Newtons, and the force grows as packaging proceeds.^49
The crystal structure of the central component of the motor from B. subtilisbacteriophage 29 illus-
trates the principle of operation of the DNA pumps (Figure 10.20a). In essence, the translocation machinery
is a rotary motor comprising a system of two rings of mismatched symmetry that interact with the DNA.50,51
The central component contains 12 copies of a 36-kDa subunit. This forms a central channel with a diameter