Hi, dear colleagues,
Recently, I really confused about ORF in plasmid. I checked my target sequence in my plasmid, in fact, after the promoter there are two possible ORFs, 1st ORF is close to my promoter, as a 56aa peptide, 2nd ORF is my target protein 219aa [CmR].
In my opinion, if the 1st ORF is translated, the 2nd ORF will keep silent and no protein production. The fact is that the 2nd ORF is translated well, the 1st ORF is silent, even it is closer to promoter. The sequence file is attached.
My questions,
Whether there is a limit for ORF length?
>In SnapGene default parameter, the ORF should bigger than 75aa, but I’m sure the short peptide could be translated. Maybe in plasmid not ??
Always the first start codon [M] could be recognized during the translation, I wonder whether the small ribosomal could choose the start codon that is included in a longer ORF. That’s really incredible, if so.
Thanks for your any help~
Yunlong
Hi Yunlong,
in bacteria the small ribosomal subunit is recruited to the mRNA through the binding of a sequence on the 16S rRNA to the so-called Shine-Dalgarno (SD) sequence, normally ending 8 nt upstream of the start codon AUG. In E. Coli the SD is AGGAGGT.
In your construct you have an almost perfect sequence, AGGAGcT, before your Chloramphenicol resistance gene and after the start codon of the small ORF. I can't say if the expression plasmid was designed in this way (with the SD actually 11 nt before the ATG, besides containing the single mutation as part of the Bpu10I RE site) or if this was the result of a cloning strategy that didn't consider this aspect of prokaryotic translation (I'm a bit surprised because the chloramphenicol sequence start is a good Kozak consensus, which is actually needed in eukaryotes...).
Anyway, it's good that your gene is translated, but again, I can't say if you might obtain higher expression with the exact SD sequence and its position correct...
Hi Yunlong!
So you have two ORFs on the same plasmid? Would it be better if you construct/obtain another plasmid with your target sequence only? In theory, both genes should be translated, the difference would be in the protein expression levels. However, it depends on the promoter as well, so I assume that the second gene (your target ORF) was eventually translated because of the earlier transcription where the promoter of the 2nd ORF was the target.
Thanks for your answers, Pietro Pilo Boyl and Yazan J Meqbil !
This sequence is retrieved from a commercial plasmid [#addgene], so I trust it as right and reasonable construction.
I am trying to understand why it works. As you said, the target gene [CmR] is translated thanks to SD sequence. It is important to recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis. Besides, an mRNA codes for multiple polypeptides in bacteria is possible. Maybe these two ORFs could be translated. One with SD sequence is highly-translated, the other w/o SD is poorly translated.
Then I checked the sequence of CmR, yes, surprisingly, Sequence is started with a good Kozak consensus. I found explanation about it, " Eukaryotic ribosomes (such as those found in retic lysate) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites. " [from https://www.thermofisher.com/fr/fr/home/references/ambion-tech-support/translation-systems/general-articles/ribosomal-binding-site-sequence-requirements.html]. It seems that SD+Kozak+ORF in palsmid is to ensure its wanted translation.
However, could I say that the SD is necessary for a prokaryotic translation plasmid ? Because it allows the bacterial ribosome to be built at an interior position on the mRNA through direct binding to this sequence.
And Kozak sequence is not necessary in an eukaryotic ORF in plasmid ? Because I found many ORFs in our plasmids express well, without Kozak sequence. Or maybe, if a strong promoter , needn't Kozak ?
Thanks again~
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