6 replies to this topic

[kevin]

Just tinkerin..

In considering how DNA polymerase requires a free 3'-OH and thus an RNA polymerase which does not is required to lay down the initial stretch of RNA nucleotides in order to kickstart DNA synthesis, I'm wondering why a DNA oriented polymerase never evolved that would be able to do the same. It would get around the linear DNA molecule shortening and telomere maintenance issue. Might we some day be able to create primase that uses DNA instead? Is there any overriding reason to have an RNA oriented primase? Is it only a relic from the "RNA world" from the time before DNA was used as the genetic material?

[kevin]

Link: http://www.ncbi.nlm....t_uids=15561142

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J Mol Biol. 2004 Dec 10;344(5):1251-63. Related Articles, Links

The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 3'-terminal nucleotidyl transferase activities.

Lao-Sirieix SH, Bell SD.

MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK.

A eukaryotic-type primase was identified in the crenarchaeon Sulfolobus solfataricus. The two-subunit DNA-dependent primase, termed PriSL, was purified following co-expression of the subunits in Escherichia coli and its activity was characterised. PriSL was capable of utilising both ribonucleotides and deoxyribonucleotides for primer synthesis in the presence of natural, or synthetic, single-stranded DNA. A broad distribution of products was detected, ranging from dinucleotides to DNA molecules in excess of 7 kb and RNA up to 1 kb in length. However, PriSL had a significantly higher affinity for ribonucleotides than for deoxyribonucleotides. Using site-directed mutagenesis, two aspartate residues crucial for nucleic acid synthesis and residues important for the binding of free nucleotides were identified. In addition to the primase and polymerase activities, we reveal that the primase possesses a template-independent 3'-terminal nucleotidyl transferase activity.

PMID: 15561142 [PubMed - indexed for MEDLINE]

[Lazarus Long]

I suspect it has more to do with the diversity of proteins and their individualized applications than as a vestigial of pre-DNA. Protein synthesis predates Eukaryotic evolution but after that combination into multicellular ability the addition of RNA synthesis would offer many more options derived of the additional molecules' binding sites.

I also suspect that as DNA is more the *core code* that its relationship with RNA allows a certain *safe* distance from overall tissue synthesis that may help preserve the integrity of that code by not depending on *exactly* the same molecular sequencing.

Remember in many respects we are more and more coming to view viruses not so much as alive as examples of the kind of rogue code threat that such a distance would help to protect against. And if I remember my theory correctly viruses didn't evolve at first but appear much later and long after bacteria.

You know me Kev I always look to think of these processes in evolutionary terms first and then try to appreciate the chemistry, though I really should go back to learn the math of the code involved. )

[kevin]

I certainly think that RNA polymerase came first and the offloading of the information function to DNA was necessary to circumvent the problems associated with the higher chemical reactivity of ribose and maintain genetic integrity. An RNA polymerase, perhaps an ancestral primase, developed a dual affinity which eventually became a single affinity for deoxynucleotides and voila, a copy of the protein producing RNA was translated into DNA whereupon RNA could assume a more dynamic role being released from the necessity of wearing both hats of information storage and protein synthesis. It would be something to find a ribozyme capable of carrying out RNA synthesis... perhaps inherent basepairing was all that was necessary in the presence of some mineral catalyst..

As one of my profs said.. just when you think you have a handle on things... you have another 3 billion base pairs to think about.. in regards to the new developments in RNA regulating information and protein synthesis concurrently and arising from what was previously erroneously known as "junk"..

Quite funny really that so many years 99 % of our DNA would be considered 'junk'

[Lazarus Long]

The closest example of autonomous RNA synthesis that I can imagine is an RNA virus but it still has to high-jack DNA to replicate.

I didn't mean to suggest that RNA polymerase wasn't around as a kind of *mineralization* process but it wasn't alive. It takes the self replication aspect to qualify IMHO.

I have heard many different theories on the origin of viruses from runaway episomes, to the kinds of environmentally available RNA polymerase that you suggest might have been present but according to the fossil record this was long after the presence of prokaryotes.

I still suspect that doing all protein synthesis with DNA would incur a kind of liability that might make DNA too vulnerable to dangerously fast mutation rates. Besides, having the step of RNA protein synthesis offers much more molecular diversity by increasing the potential number of amino acids involved along with a more *recyclable* molecule for the cell to utilize.

I think of DNA as code and RNA polymerase as a kind of super *catalyst* that expresses the code in combination with amino acids and other molecules.

[John Schloendorn]

It is thought that the requirement for a free 3' OH group is part of the proofreading activity that all non-viral DNA Polymerases have. The DNA-Polymerases check whether the previous base pair is correct, before they pair the next one, (and hydrolyze it off, when it is not) which gives them some additional 3 orders of magnitude accuracy. When there is no previous base-pair to check (i.e. 3' OH), they can't operate. (Remember the RNA primers are later removed and replaced by DNA that restores accuracy. And that's really the end-replication problem.)
RNA polymerases don't have the proofreading function because they don't need to be as accurate. (Each DNA molecule makes many more Proteins than each RNA molecule) Thus, they also don't need primers.

I can't think of any reason that this would be compulsory once we do the engineering bit. But as for telomere maintenance as a near-term anti-aging strategy... I have my doubts.

[kevin]

I understand the requirement for higher accuracy with DNA synthesis but actually RNA Polymerase II at least, although much less accurate than DNA PolIII, does have a 3'->5' exonuclease activity for hydrolyzing mismatched base pairs. It's amazing how prevalent the notion that RNAPol's have no proofreading.. even my cellular biology profs say it this way although we learnt in biochem that this is not so... at least with RNAPol II.. I'm not sure at the moment if it exists in I or III.

Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS
(RNA-DNA dumbbell / transcriptional proofreading)

ChoonJu Jeon and Kan Agarwal*

Fidelity of DNA and protein synthesis is regulated by a proofreading mechanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its control has been hampered by the necessity to employ complex DNA templates requiring either a promoter and initiation factors or 3-extended templates. To circumvent this difficulty, we have created an RNA-DNA dumbbell template that can be recognized as a template-primer and extended by RNA polymerase II. By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out template-dependent elongation at the mispaired end. The transcripts containing misincorporated residues can be cleaved by the very slow 3  5 ribonuclease activity of the RNA polymerase II, but enhancement of this activity by the elongation factor TFIIS generates RNA with a high degree of fidelity. This enhanced preferential cleavage of misincorporated transcripts suggests an important role for TFIIS in maintaining transcriptional fidelity.

http://www.pnas.org/...ull/93/24/13677

I don't see telomere maintenance as being that relevant to anti-aging or at least it likely plays only a small part in the overall picture. I just wonder if the reason telomerase and the current system of telomere maintenance even arose was because of the "choice" of RNA primer vs DNA and wonder if there's some underlying critical reason why a DNA Pol has never evolved to replace that functionality.