Silly question, fidelity of RNA polymerase - transcription is 100% error free ? (Oct/28/2006 )
well, I don't know is that too silly to ask this question
we all know DNA polymerase do make some error in PCR, but,
how about RNA polymerase ? I haven't heard about that.
I don't think this enzyme can be totally error free, right ?
and that means protein being overexpressed (or normally expressed in organism) would contain some "mutant" ?
may be 1 million molecule contain only one "mutant" and cannot be detected.....however, would that be a problem in molecule screening, for example, ribosome-display screening?
RNA polymerase makes errors too.
But effects are relatively minimized by the fact a non sense RNA may be degraded faster and the fact the true half life of an mRNA is very short.
If you consider the RNAs that have longer half life, like pol III ones, the copy number of them is so great that you have plenty fully functional ones and that drive the uncorrect one negligible.
The commercially available reverse transcriptases produced by Promega are quoted by their manuals as having error rates in the range of 1 in 17,000 bases for AMV and 1 in 30,000 bases for M-MLV.
we all know DNA polymerase do make some error in PCR, but,
how about RNA polymerase ? I haven't heard about that.
I don't think this enzyme can be totally error free, right ?
and that means protein being overexpressed (or normally expressed in organism) would contain some "mutant" ?
may be 1 million molecule contain only one "mutant" and cannot be detected.....however, would that be a problem in molecule screening, for example, ribosome-display screening?
RNA polymerase is not the same as reverse transcriptase. RNA polymerase is not used in PCR, to my knowledge. RNA polymerase has a fidelity about 10 fold worse than DNA polymerase (wild type E coli RNA pol compared to any of the wild type E coli DNA pols.)
I don't know the fidelity of reverse transcriptase. Given the life cycle and life style of a retrovirus, I would assume that revese transcroptase has a lower fidelity (more errrors) than DNA pol.
Of course, the commercially available DNA pol Taq's have varying degrees of fidelity.
If you mean RT enzyme's, they have higher error rates than DNA polymerases, (taq, pfu, any other). Recently, stratagene has released a new one with lower error rate: http://www.stratagene.com/products/display...ct.aspx?pid=700.
Don't know if it's any good.
If you consider RT-PCR, you indeed have the possibility of inserting an error during cDNA synthesis, but during the PCR itself you have 35 cycles during which you can "create errors".
So, if you RT-PCR for sequencing, consider the fact that on a population level, you won't see too much RT or PCR errors, but on a clonal basis you will have errors, brought in during PCR or RT steps.
(btw: retroviruses depend on their error prone reverse transcription for survival, estimates of their error rates vary from about 1/5000 or so to 1/30000).
Actually, I am talking about RNA polymerase not reverse transcriptases
I know they are different, former is from DNA to RNA and the latter is from RNA to DNA
I just wonder, in normal protein expression, is there any error occur ? and the protein obtained is not the thing I want indeed. 
There should be errors occur in normal protein expression, but abnormal protein amounts to very very little compare to normal protein, and can be ignored (or you could not detect it). I think
and do remember abnormal/misfolded proteins are targeted for degradation.
And in eukaryotes, there is a RNA monitoring system, that degrades mRNA which have premature stop codons within the transcript.
I know they are different, former is from DNA to RNA and the latter is from RNA to DNA
I just wonder, in normal protein expression, is there any error occur ? and the protein obtained is not the thing I want indeed.
Yes, there is a lot of error; in a wild type scenario there is about 10 fold more error in transcription than in replication.
I believe this is because DNA pol has a very good proofreading function.
Higher errors are tolerated in transcription because a few bad transcripts out of thousands of good ones, will not kill off a cell. In replication, a mistake can be fatal to the daughter cell.
But why did you mention PCR, in this case? RNA pol has very little to do with PCR. Are you thinking about the mRNA that you might harvest for use as a template in RTPCR?
-Patty, an old transcription person
Actually, I am talking about RNA polymerase not reverse transcriptases
I know they are different, former is from DNA to RNA and the latter is from RNA to DNA
I just wonder, in normal protein expression, is there any error occur ? and the protein obtained is not the thing I want indeed.
Yes, there is a lot of error; in a wild type scenario there is about 10 fold more error in transcription than in replication.
I believe this is because DNA pol has a very good proofreading function.
Higher errors are tolerated in transcription because a few bad transcripts out of thousands of good ones, will not kill off a cell. In replication, a mistake can be fatal to the daughter cell.
But why did you mention PCR, in this case? RNA pol has very little to do with PCR. Are you thinking about the mRNA that you might harvest for use as a template in RTPCR?
-Patty, an old transcription person
Thank,
and why I mention PCR is because, they are both polymerase
I just afraid of it ---- Recombinant protein which is used for medical use and should be injected into blood. Then, if those protein being expressed by bacteria contain significant amount of toxic protein, say, 0.1%, it should be a problem.
One of a lecture in our department is doing a project like this, therefore, I want to know the error rate of RNA polymerase in bacteria.
