Good day.
I was wondering whether it is allowed to have stop codons in the 5' leader sequence... On the one hand, the eukaryotic ribosome falls off as soon as it hits a stop codon. Well, it scans along the mRNA from the 5' cap until it meets a kozak-ATG where it starts translating...
My 5'UTR would have this sequence agatcTAAgccaccATG...Is that a problem?
pFlag-cmv10 has GCTCGTTTAGTGAACCGTCAGAATTAaccATG as the leader sequence so a frame-shifted taa seems to be ok... Maybe also the position of taa is important?
Thanks a lot!
The reading frame and with it the codons of any coding sequence of a given gene is defined by the translational start codon. upstream of the first coding Met there is no reading frame and with it no stop or any other codons. Some 5' UTR have ATGs upstream to the translational start site, this is not uncommon but those are not recognized by the ribosome.
Hi there,
Indeed the purpose of the scanning step of 5' region of mRNA by the small subunit of the ribosome is to recognize/choose the initiation codon for translation. Whatever the sequence of the 5'UTR and its content in stop codon, the assembly of the ribosome will occur only when ATG is reached by the small subunit. The role of stop codon in a sequence is essential only during effective translation.
I would worry more about upstream ATGs than upstream stop codons. ATG could set the frame and if the ribosome prefers to use it as a start codon (Kozak consensus sequence notwithstanding), your intended ATG may be completely missed. Verify your protein expression by western blot using an anti-FLAG antibody (and an antibody against your tagged protein) and make sure your protein is the proper predicted size. That should validate that the ATG in your FLAG-tag set the frame and your downstream sequence is expressed. It's good to check that you are in-frame by sequencing your construct beforehand (especially if your insert was a PCR product).
Thanks for all the answers!
http://en.wikipedia.org/wiki/Eukaryotic_translation#/media/File:Eukaryotic_Translation_Initiation.png
For what I understand from this picture is that only the small subunit scans along the mRNA and when finding the ATG, the large subunit assembles on it. The ribosome disassembles at stop codons, but it can't disassemble when it isn't assembled yet. Right?
No assembly, no disassembly. If a startcodon is not encountered by the scanning 43S, NMD kicks in and removes the "m"RNA, or if in mitosis, recognizes it as an lncRNA for nuclear re-import after completed cell division.
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