Primer strand

Unlike what happens in DNA replication, where both strands are copied, only one of the two DNA strands is transcribed into mRNA. The strand that contains the gene is often called the coding strand, or primer strand, and the strand that gets transcribed is called the template strand. Because the template strand and the coding strand are complementary, and because the template strand and the transcribed RNA are also complementary, the RNA )no ecule produced during transcription is a copy of the DNA coding strand. The only difference is that the RNA molecule has a U everywhere the DNA coding strand has a T. 

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Exonuclease activities, proofreading, and editing. DNA polymerase I not only catalyzes the growth of DNA chains at the 3 end of a primer strand but also, at about a 10-fold slower rate, the hydrolytic removal of nucleotides from the 3 end (31- 5 exonuclease activity). The same enzyme also catalyzes hydrolytic removal of nucleotides from the 5 end of DNA chains. This latter 5 - 3 exonuclease activity, the DNA polymerase activity, and the 3 -5 exonuclease activity all arise from separate active sites in the protein. DNA polymerases II and III do not catalyze... 

The discovery of DNA polymerase and its dependence on a DNA template led to the search for enzymes which could make an RNA molecule complementary to the DNA. RNA synthesis does not require a primer strand it does, however, require a specific initiation signal on the DNA template strand to allow binding and initiation. As the RNA strand is synthesised it forms a temporary helix with the template DNA, but when complete the mRNA breaks off at the stop site on DNA. Once released from DNA, some of the RNA is processed further, for the specific structures of rRNA and tRNA. 

DNA polymerases catalyze DNA synthesis in a template-directed manner (Box 16). For most known DNA polymerases a short DNA strand hybridized to the template strand is required to serve as a primer for initiation of DNA synthesis. Nascent DNA synthesis is promoted by DNA polymerases by catalysis of nucleophilic attack of the 3 -hydroxyl group of the 3 -terminal nucleotide of the primer strand on the a-phosphate of an incoming nucleoside triphosphate (dNTP), leading to substitution of pyrophosphate. This phosphoryl transfer step is promoted by two magnesium ions that stabilize a pentacoordinated transition state by complex-ation of the phosphate groups and essential carboxylate moieties in the active site (Figure 4.1.1) [2],... 

Fig. 2.1. Chain extension reaction catalysed by DNA polymerase I. Chain growth occurs in the 5 - 3 direction by the stepwise addition of nucleotides to the free 3 -OH end of the primer strand.

The primers used are restriction fragments which originated from within the duplex DNA to be sequenced. In the annealing reaction, to produce the primer-template complex, only one of the primer strands is hybridized to its complementary sequence in the template since the other potential template is destroyed by the Exo III treatment. The result is the formation of a primer-template which can be extended by DNA polymerase in a direction opposite to that of the Exonuclease III attack. This is shown in Fig. 3.15. Two inherent features of the method which can cause problems are also made clear in Fig. 3.15. In the first place priming and chain extension can also occur on the 3 -ends of both template strands when sufficient complementarity remains between the 3 -ends of the template strands to form a base-paired structure. Secondly, a primer which originates from near the centre of the duplex DNA... 

Enzymatic replication of DNA is a complex process and requires the cooperation of some 20 or more proteins. Arthur Komberg and his colleagues first discovered an enzyme in E. coli that catalyzed the polymerization of deoxyribonucleotides under the direction of a DNA template. This enzyme, DNA polymerase I (Pol I), is now known to be only one of a group of similar enzymes that can copy DNA or RNA templates or both. Three distinct enzymes (Pol I, Pol II, and Pol III) have been isolated from bacterial cells (Table 11.1). These enzymes catalyze the stepwise addition of deoxyribonucleotide residues to the free 3 -hydroxyl end of a preexisting DNA or RNA primer strand thus the enzymatic elongation proceeds in the 5 —> 3 direction. The overall reaction is... 

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