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Enzyme Evolution:
Recent scientific studies provide strong evidence that nucleic acid polymerases share a common ancestor. Structural analyses support the notion that the similarities between polymerases go beyond the arbitrary assignment into classes on the basis of the use of DNA or RNA templates and deoxyribonucleoside or ribonucleoside triphosphates (Figure 18). In fact, it has been postulated that most of the nucleic acid polymerizes belong to a polymerase superfamily containing closely related active sites that are similarly positioned within the polymerase active cleft (Figure 19). Thus, it appears that there is a genetic polymerase module that provide the active site the architecture to carry out the phosphoryl transfer reaction (Joyce, 1997). Only subtle modifications to this module achieve the substrate specificity that is unique for each polymerase class. We see over and over in the course of evolution the tendency of nature to copy and paste particular structural motifs. Figure 18.jpg (65987 bytes)Figure 18.
Schematic representation illustrating the division into classes of nucleic acid polymerases in terms of sugar and template specificities.
As Joyce C. (1997) has stated "nucleic acid polymerases are suffering from an identify crisis." Recent published papers have brought us, a DNA polymerase as a reverse trancriptase (Ricchetti et. al., 1993), the T7 RNA polymerase as a DNA polymerase (Sousa el. al., 1995), and a DNA polymerase as an RNA polymerase (Gao et. al.,1997). This switch in specificity was done by single amino acid substitution. As illustrated in Figure 19, the polymerase domains of the Klenow fragment, the HIV-1 RT, and that for T7 RNA polymerase are structurally similar. The yellow spheres shown in the palm domain represent the catalytic aspartate residues. The relative positions of the residues involved in sugar discrimination are indicated in each polymerase structure. Figure 19.jpg (144703 bytes)
Figure 19. Illustration of the polymerase domains of Klenow fragment, HIV-1 RT, and T7 RNA polymerase (Joyce, 1997).
DNA polymerases discriminates against ribonucleotide through a "steric gate" mechanism. For example, the Klenow fragment and the HIV-RT polymerase discriminate against ribose sugar because bulky amino acid residues (Phe762 and Tyr155, respectively) make steric interaction with the 2'-OH group of the incoming nucleotide (see Figure 19). The Moloney murine leukemia virus (MoMLV) polymerase contains Phe155, which is homolog to Tyr155 in HIV-RT. Figure 20 shows how Phe155 in MoMLV clashes with the 2'-OH group of the incoming ribonucleotide (Gao et. al., 1997). Thus, mutation of this bulky residue to alanine, as it was found, will switch the sugar specificity of the polymerase. Figure 20.jpg (90970 bytes)
Figure 20. In MoMLV RT, Phe155 acts as a "steric gate" to prevent incorporation of r-NTPs (Gao et. al., 1997).
On the other hand, T7 RNA polymerase uses a totally different mechanism to discriminate against deoxyribose nucleotides. Studies have shown that, in T7 RNA polymerase, Tyr639 makes hydrogen-bonding interaction with the 2'-OH group of the incoming ribonucleotide. Mutation in this position from Tyr to Phe renders the polymerase specific for deoxynucleotides. (Sousa et. al., 1995).
Bacteriophage T7 RNA polymerase shares some amino acid sequence similarities with other RNA polymerases. Table 2 illustrates the result obtained when the FASTA program was used for sequence alignment. As it is shown, only bacteriophage T3 RNA polymerase has the highest amino acid identity (82 %) to T7 RNA polymerase, while the other RNA polymerases share only about a 29 % amino acid identity.Figure 21. Multiple alignments correspoding to part of the polymerase domain of T7 RNA polymerase, 1QLN, and other RNA polymerases. In (A), the concensus domain among RNA polymerases is shown relative to the amino acid sequence of T7 RNA polymerase; in (B), the alignment sequence is displayed. The polymerases are labeled according to their PID accession number.