410 Chapter 10
Figure 10.16 (a) A composite of DNA packaging proteins from the three domains of life. Comparison of the archaeal
and eukaryotic histones (PDB: 1F1E, 1BFM and 1AOI). (b) Interaction of the SUL7d protein in the
minor groove of DNA
resulting in attack on the -phosphate of the incoming nucleoside triphosphate. A second metal ion helps
to orient the -phosphate of the nucleotide triphosphate, so as to stabilize the penta-covalent transition state,
and to assist the departure of the pyrophosphate product from the catalytic site. The proposed two-metal ion
mechanism is based on both the DNA polymerase and the 3–5exonuclease domains of DNA polymerase
I, the latter of which serves a proofreading function to ensure fidelity in the replication of its DNA.
The DNA polymerases must have no specificity for particular DNA sequences, but they must be spe-
cific for correct Watson–Crick base pairing, for example as seen in the interaction of T7 DNA polymerase
with DNA (Figure 10.17a). Studies of replication of DNA with nucleotides having chemically modified
bases suggest that the fidelity is due largely to steric exclusion and shape-matching.^44 Mismatches are also
discriminated by contacts to the minor groove of the DNA. Conformational changes are necessary to adapt
the protein to the substrate during the polymerisation, and the polymerases have a striking modular organ-
ization in which different portions undergo movement, much like a right handthat grasps and manipulates
substrates and translocates products (Figure 10.17b). Accordingly, these domains have been labelled ‘thumb’,
‘palm’ and ‘fingers’.
10.7.2 DNA-Directed RNA Polymerases
The DNA-directed RNA polymerases(E.C. 2.7.7.6) contain a 400-kDa catalytic core that has a con-
served structure and function in all three domains of life. RNA polymerases do not recognize or bind
avidly to the promoter DNA themselves. Instead, they require the help of auxiliary proteins that recognize
both the target and recruit the enzyme. Nearly a dozen basal transcription factors are involved in the initi-
ation of transcription in eukaryotes; whereas in bacteria, things are much simpler, and initiation involves
only one ‘ -factor’ protein, of which there are many different types.
The RNA polymerases have an initiation and elongation phase in transcription. During the initiation
phase, the polymerase binds to a promoter sequence, melts the DNA to form a transcription ‘bubble’ and