what reading frame? - (Feb/02/2014 )
Hallo, a rookie question, but how do I know what the reading frame of a plasmid is?
I have the sequence of a plasmid. It contains a promotor sequence and after this sequence there is a restriction site and right after this restriction site there is the atg sequence. Does that mean this atg is the right start?
Or can the start codon also be one of the atg codons in the promotor?
Usually what happens is that the promoter modifies how often something is transcribed and includes a RNA polymerase binding site. From this site, the RNA pol works its way along the sequence until it reaches an ATG site, where it will start transcription. The promoter does not need to be in-frame with the ATG.
bob1 on Sun Feb 2 21:43:47 2014 said:
Usually what happens is that the promoter modifies how often something is transcribed and includes a RNA polymerase binding site. From this site, the RNA pol works its way along the sequence until it reaches an ATG site, where it will start transcription. The promoter does not need to be in-frame with the ATG.
Ok thanks.
A second question: its a plasmid that contains 3 genes in a row (so they are all , or should be, transcribed/translated as 1 gene). Now I wonder: the first ATG is the start, but there are some ATG codons in the entire gene after the first one... Will this not hinder the transcription/translation?
And what with stop codons? I did find some in the sequence, so I wonder how this can work.
The polymerase will elongate the transcript from the ATG start to the polyA signal site (usually consensus sequence AATAAA) where it detaches and adds many As to the 3' end of mRNA.
Ribosome binds somewhere before the ATG and translates codons (where ATG encodes for ordinary methinonine inside the mRNA strand and doesn't have any other function) until they reach a stop codon, after which it detaches.
If you want to translate more genes from the same ATG (so theay are polycistronic, all on the same mRNA molecule) you need to have internal ribosomal binding site (IRES) between them so the ribosome will reattach. Without it ribosome can't go over stop codon.
Or the genes need to have their own promoter.
Alternative way of creating more proteins from one mRNA is inclusion of 2A peptide self-cleaving sequence between the genes, single protein is created (the original genes must not have the stop codon!) and then cleaved at 2A sites.
If your genes are not separated by anything and don't even have a stop codon in the middle, in that case they are 'fused', they will create single hybrid protein (and in that case I wouldn't probably call them 'genes', for clarity). You should know what exact type of plasmid you have.
Trof on Mon Feb 3 10:32:47 2014 said:
The polymerase will elongate the transcript from the ATG start to the polyA signal site (usually consensus sequence AATAAA) where it detaches and adds many As to the 3' end of mRNA.
Ribosome binds somewhere before the ATG and translates codons (where ATG encodes for ordinary methinonine inside the mRNA strand and doesn't have any other function) until they reach a stop codon, after which it detaches.
If you want to translate more genes from the same ATG (so theay are polycistronic, all on the same mRNA molecule) you need to have internal ribosomal binding site (IRES) between them so the ribosome will reattach. Without it ribosome can't go over stop codon.
Or the genes need to have their own promoter.
Alternative way of creating more proteins from one mRNA is inclusion of 2A peptide self-cleaving sequence between the genes, single protein is created (the original genes must not have the stop codon!) and then cleaved at 2A sites.
If your genes are not separated by anything and don't even have a stop codon in the middle, in that case they are 'fused', they will create single hybrid protein (and in that case I wouldn't probably call them 'genes', for clarity). You should know what exact type of plasmid you have.
I think its indeed more the last one: its a plasmid with a promoter, tags for immunoprecipitation, gene of interest and fluorophore. And they are linked with glycine linkers.
So I assume its just 1 promoter for all the "genes" and 1 stop codon at the end.
But when I look look at the sequence I find multiple "stop" codons in these "genes".
This is just confusing.
an extra question: is it possible to have different reading frames in a plasmid working at the same time?
I noticed that the start codon of one of the antibiotic resistant markers uses another reading frame than the start codon of the tags for immunoprecipitation , the gene of interest and the fluorophore.
So you actually have "a gene" with a N-tag (possibly Myc tag or else) and C-tag (GFP or similar) transalted together. Glycine is just used to separate the domains.
But if you have stop codons within the sequence, it's not quite right. There are specific situations, where there is a way how to overcome stop codon, for some special purpose as I recall, but you should know if that's the case.
Where are the stop codons? Are on the expected ends of the each "gene" transcript" or somewhere in the middle?
Can you locate the polyA signal?
All transcription happens relative to polymerase bindig site (i.e. the promoter). No other relative possition is relevant. You may have several reading frames on a same DNA strand (of even on the opossite) that are off-frame to each other, but every one is "inframe" to its respective promoter. Or as was said, there just needs to be a ATG in the vicinity of the promoter and polymerase will find it, if there are more ATGs close by, probably transcription will start from either of them. But that's usually not very desired, because it has no regulation over that, so if in a living organism there are supposed to be alternate reading frames within one sequence, I would expect each of them to have it's own promoter.
Yes,
its indeed a gene with an N-tag and C-tag!
I checked again for the stop codons, I can find them, but they seem to be in a different reading frame (I used the reading frame that correspondends with the ATG start codon right after the promoter).
This seems ok.
The poly A tail, no I could not find that.
I used your sequence: AATAAA , perhaps its another one?
(I found AATAAA, but in another reading frame than the ATG start codon right after the promoter).
Trof on Mon Feb 3 12:16:44 2014 said:
So you actually have "a gene" with a N-tag (possibly Myc tag or else) and C-tag (GFP or similar) transalted together. Glycine is just used to separate the domains.
But if you have stop codons within the sequence, it's not quite right. There are specific situations, where there is a way how to overcome stop codon, for some special purpose as I recall, but you should know if that's the case.
Where are the stop codons? Are on the expected ends of the each "gene" transcript" or somewhere in the middle?
Can you locate the polyA signal?
All transcription happens relative to polymerase bindig site (i.e. the promoter). No other relative possition is relevant. You may have several reading frames on a same DNA strand (of even on the opossite) that are off-frame to each other, but every one is "inframe" to its respective promoter. Or as was said, there just needs to be a ATG in the vicinity of the promoter and polymerase will find it, if there are more ATGs close by, probably transcription will start from either of them. But that's usually not very desired, because it has no regulation over that, so if in a living organism there are supposed to be alternate reading frames within one sequence, I would expect each of them to have it's own promoter.
Poly-A signal doesn't need to be inframe. The frame only matters from ATG to stop codon, after that, there is the 3' untranslated region within which there has to be poly-A signal to physically end the template-dependent polymerization.
So if your AATAAA is after the stop codon of the C-tag, it's your poly A signal.
(you will not find "tail" in DNA, the tail is only produced on the end of the mRNA as the result of the AATAAA signal sequence and it's template-independent)
Oh ok.
After the "genes" (the N tag, "gene" and C tag) there is a terminator (this terminator contains many stop codons ... I guess this terminator is also "stop codon", I mean: the first stop codon (three letters, TAG,TAA or TGA) is to be found in this terminator sequence, not really right after the end of my "c-tag", meaning after the C-tag( the fluorophore) there are still a few 3 codons left.
Is this normal or do people normally put a stop codon (for example TAG) right after the tag sequence ? And is it normal to have a terminator sequence all the time of is the stop codon enough?
I guess it all depends on what you want?
Trof on Mon Feb 3 12:54:45 2014 said:
Poly-A signal doesn't need to be inframe. The frame only matters from ATG to stop codon, after that, there is the 3' untranslated region within which there has to be poly-A signal to physically end the template-dependent polymerization.
So if your AATAAA is after the stop codon of the C-tag, it's your poly A signal.
(you will not find "tail" in DNA, the tail is only produced on the end of the mRNA as the result of the AATAAA signal sequence and it's template-independent)
For ribosome, only one and first stop codon matters.
Imagine you're reading a book, until you get to a command saying "now stop reading and go away". And you do. So eventhough there may be furter text in the book, of furter commands saying you to do something, it doesn't matter, because you already stopped and left. That's what ribosome does.
The sequence after THE stop codon is kind of "junk" (in fact it most likely is NOT useless in the organisms, beacuse it may contain regulatory sequences, that may affect translation rate or bind other factors doing various things), but it's not important for he ribosomal elongation of the protein.
Terminator contains stop codon (possibly for every frame there is, just to be sure), poly A signal for the respective type of organism (prokaryotic, eukaryotic). Some may contain expression enhancers. This is just a common building brick of plasmids.
As for the C-tags, there doesn't need to be end exactly after the functional protein domain. There can be some "dummy" amino acids, if they don't affect the function of the tag, they doesn't matter.