Chemistry Reference
In-Depth Information
acid sequence of RNA polymerases, NA
D
F
D
G
D
, is conserved among RNA
polymerases from bacteria to high eukaryotes.
38
This motif is located on a loop con-
necting two strands in a b-barrel domain, as observed in Taq and yeast RNA pol
II
38,39
(Figure 15.3C). This forms the third class of polymerase catalytic centres.
Regardless of the differences in the tertiary structure and catalytic residue
locations, all three classes of DNA and RNA polymerases appear to require two
metal ions in the active site for substrate binding and the phosphoryl transfer reac-
tion. In the best-studied DNA polymerase, pol b (X family), the two Mg
2+
ions are
jointly coordinated by two conserved Asps and the a - phosphate of an incoming
dNTP
30
(Figure 15.3B). Both Mg
2+
are coordinated by six ligands in the octahedral
geometry. One metal ion (conventionally known as the A metal ion) is close to the
3
-OH of the primer strand and suspected to activate it for nucleophilic attack, and
the other metal ion (known as the B metal ion) is coordinated by the b and g phos-
phates (the leaving group) and is suspected to facilitate product formation. The
metal ions are separated by
′
3.4 Å
in the enzyme transition-state complex.
40
perhaps drawn close together by the tran-
sitional highly negatively charged phosphate anion. Perhaps not surprisingly, the
same metal ion requirement and coordination geometry have been observed in pol
l and m of the same X family
30
and in the related CCA-adding enzymes.
37
Interestingly, similar coordination of the two metal ions have been observed in
pol I-like polymerases, including T7, Taq and Bst DNA pol of the A family,
41 - 43
RB69
DNA pol of the B family,
44
HIV RT,
45
and Dpo4, pol k and Rev1 of the Y family.
31
For the RNA pol II-like catalytic centre, in the currently available structures of
RNA pol II complexed with a nucleic acid substrate, the metal ions are not coordi-
nated as in DNA pol I or pol b, and are further apart than 4 Å. This may be due to
the double conformations of the incoming UTP
46
or the absence of the 3
∼
4 Å in the enzyme-substrate complex and by
∼
- OH in
the RNA strand.
47
Given the three conserved Asp residues in the active site, it is
likely that these carboxylates and the a-phosphate of the incoming NTP jointly
coordinate the two Mg
2+
ions for catalysis as with the other two classes of
polymerases.
′
15.2.1 Alignment of Two Metal Ions and Fidelity of DNA Synthesis
Displacement of two metal ions from the canonical binding sites in fact has been
observed in the active site of several DNA polymerases. For example, Dpo4, which
is capable of translesion synthesis, has a large, preformed and solvent-exposed active
site.
48
An omission of the 3
-OH in the primer strand, which is commonly used to
crystallize polymerase-substrate ternary complexes, leads to
′
1 Å displacement of
metal ion A.
41,47,49,50
Although a variety of modifi ed template bases (lesions) and
mismatched replicating base pairs can bind in the active site of Dpo4,
48
the catalytic
effi ciency differs with different substrates, and effi cient nucleotide incorporation
appears to correlate with the canonical confi guration of two metal ions.
50
Similar to
Dpo4, DNA pol l also has a preformed active site, and one of the two metal ions
is frequently missing from the active site in correlation with the misalignment of
reaction groups in the crystal structures.
51,52
Such correlation between the metal ion
∼
