Dear Tafrihi,

A very intriguing question! DNAP can only catalyse the SN2 nucelophilic attack of 3' OH to the alpha phosphate of the incoming complementary nucleotide. So it needs a primer-template junction to begin adding the dNTPs to the new strand. But, it cannot add the first nucleotide where it has to hold on to the cognate dNTP and then add the next nucleotide to it. Whereas, RNAP takes the first NTP (which remains as NTP as there is no nucleophilic attack on it) holds on to it and then adds the 2nd and subsequently all the nucleotides to it. This difference makes DNAP a very specific enzyme which can only polymerize or extend an existing chain. This probably gives DNAP an evolutionary advantage, as most misincorporation of nucleotides tend to occur in the initial stage where the base pairing between the new strand and template strand is less, so if there is a RNA primer initially, which is of sufficient length, then there is lesser chances of misincorporation. Later the primers are replaced by DNAP I. But for RNA synthesis, that high fidelity isn't required, so RNAP is allowed to start de novo. But any such errors by DNAP at the initial stage will slow down the replication progression, which is avoided by the primers incorporated by primase and later carefully replaced by DNAP. I think this could be one the reasons why DNAP can't initiate de novo synthesis while RNAP can.

Hi Majid,

The free 3'-OH  is the one who does the nucleophilic attack on the alpha-Phosphate to synthesize DNA. On the other hand RNA polymerase does not need a free 3'-OH because it has the capability by itself to bond to ribonucleotides and generate an RNA chain de novo.

Hope it helps. Regards,

Marc

I had the same doubt of Majid Tafrihi. Nucleophilic attack was the basic theory explained in books, but is there any explanation regarding structural difference between the DNA polymerase and RNA polymerase with respect to the requirement of free 3'hydroxyl group

As far as I am concerned the ω subunit is the responsible of  facilitating assembly of RNAP and stabilizing the assembled RNAP. However I don't know which reactions are involved in this de Novo synthesis.

Regards

Hi there,

Actually the chemistry of polymerization is the strictly the same for DNA and RNA. What makes the difference is actually the specificity of RNA and DNA polymerases. RNA polymerase is able to start from a naked single stranded DNA template whereas DNA polymerase requires a partially double stranded structure exhibiting a free 3'OH and 5' overhang as a single stranded matrix to copy.

Dear Tafrihi,

A very intriguing question! DNAP can only catalyse the SN2 nucelophilic attack of 3' OH to the alpha phosphate of the incoming complementary nucleotide. So it needs a primer-template junction to begin adding the dNTPs to the new strand. But, it cannot add the first nucleotide where it has to hold on to the cognate dNTP and then add the next nucleotide to it. Whereas, RNAP takes the first NTP (which remains as NTP as there is no nucleophilic attack on it) holds on to it and then adds the 2nd and subsequently all the nucleotides to it. This difference makes DNAP a very specific enzyme which can only polymerize or extend an existing chain. This probably gives DNAP an evolutionary advantage, as most misincorporation of nucleotides tend to occur in the initial stage where the base pairing between the new strand and template strand is less, so if there is a RNA primer initially, which is of sufficient length, then there is lesser chances of misincorporation. Later the primers are replaced by DNAP I. But for RNA synthesis, that high fidelity isn't required, so RNAP is allowed to start de novo. But any such errors by DNAP at the initial stage will slow down the replication progression, which is avoided by the primers incorporated by primase and later carefully replaced by DNAP. I think this could be one the reasons why DNAP can't initiate de novo synthesis while RNAP can.

DNA polymerases are template dependent while RNA polymerase can act template independent.

Dear Amit,

I have heard of only the RNAP which adds CCA to the 3' of tRNA, the CCA adding enzyme to be the only template independent RNAP. Can you give me some other examples? Can we decide the basis of evolution with respect to this enzyme solely? All other RNAP which synthesizes the major percentage of cellular RNA, require templates.

Yes, very interesting question indeed. Of course, if this were the case we would not need telomerase and the Hayflick limit would be irrelevant. DNA replication would be more efficient as well. So why? It is accepted that RNA did evolve before DNA, and that the lack of 2'-OH in DNA may make it more stable. But, are RNA primers just evolutionary relics of our RNA days?

In 2005, a group at the NIH (Iyer L.M., et al. Nucleic Acids Res. 2005) used bioinformatics to identify human PriPol. This archaea-eukaryotic primase has the ability for de novo synthesis of DNA primers and has DNA polymerase activity. So they do exist. And they are needed for DNA replication, especially on damaged templates (Bianchi et al. Mol. Cell 2013). Regardless, DNA replication initiation and Okazaki fragment formation still requires RNA primer synthesis - our still need telomerase because of this. Do we just need more time to evolve and streamline our cells to use DNA primers instead of RNA and save energy. Or are RNA primers serving another purpose? Great question, thanks!

I think to answer this question we must pay attention to the structure of the active site of the enzyme. I mean the amino acid sequence and/or cooperation of metal ions.

good explanation by Tias saha.... dear Derek J Hoelz your statement  "Do we just need more time to evolve and streamline our cells to use DNA primers instead of RNA and save energy" it was excellent analysis which is an addition to the Tias Saha's DNA pol more cautious in replication. 

Hi there

DNA polymerases add nucleotides to the 3′ end of a polynucleotide chain. The polymerase catalyzes the nucleophilic attack of the 3′-hydroxyl group terminus of the polynucleotide chain on the α-phosphate group of the nucleoside triphosphate to be added. To initiate this reaction, DNA polymerases require a primer with a free 3′-hydroxyl group already base-paired to the template. They cannot start from scratch by adding nucleotides to a free single-stranded DNA template. RNA polymerase, in contrast, can initiate RNA synthesis without a primer

@Ammar Mendel

We already discussed that. The question is why DNAP cannot start de novo whereas RNAP can. Please read the above answers.

A point that has been mentioned but not developed is RNA polymerase's ability to bind to or "hold on to" (I'm quoting Tia Saha's post) an initial NTP as the RNA polymerase catalyzes that NTP's connection to a second NTP. This is how the first two nucleotides in the RNA primer are created, it would seem.

So as an attempt to develop that point just a bit, let me tell you two things I don't know:

1) Does RNA polymerase bind to just the first NTP, or can it also make a connection to the template strand of DNA, thus holding the first NTP on that strand as the RNA polymerase does its work?

2) Something that would make everything more convenient would be if any NTP at all could be placed on the template strand and held in place there by just the hydrogen bond between its nitrogenous base and the nitrogenous base of the first nucleotide of the template strand. This would enable the RNA polymerase to "keep its hands free" to attach the second NTP to the first one.

The order of bonds established could be a) the first NTP being hydrogen-bonded to the template strand, then b) the second NTP being hydrogen-bonded to the template strand, and finally c) the establishment of the phosphodiester bond between the NTPs. I wonder why this approach isn't feasible.