can transcription happen in cytoplasm? - This question had been posted in the General Biology Discussion board (Jan/02/2007 )
When transfecting plasmid DNA into cells, where does the mRNA being trancribe? I did a reporter assay of beta-catenin with the TOPFLASH (TCF/LEF binding site with a TA promoter driving luciferase gene), I had been challenged that the the transactivation of the luciferase gene can happen in the cytoplasm. Does this means that the transcription of the reporter construct along with the translation all happened in the cytoplasm? Because in my mind the transcription always happen in the nucleus whether or not it is a transient transfection of plasmid DNA or a stable gene in the genome. Can anyone give any clue and a reference would be appreciated. Thank you and happy new year to all !!
i think if transcription factors and other requirements needed are available in the cytoplasm, why not...but don't forget proteins and enzymes necessary for post-transcription..
Theoretically I'd agree with strawberry. I had the same concern for quite some time now. In the SIVB conference in June in Minneapolis, a system was presented showing "nano-spikes" where cells were impaled. When DNA was tethered to the tip of the spike, transcription was observed when the nucleus was impaled and I remember the guy presenting said that the nano-pike had to go through the nucleus. That was on animal cells.
My personal experience is with plant cells. I also use nanocarriers to deliver DNA inside the cell. The nanocarriers clearly remain in the cytoplasm but transgene expression (GFP) is observed very clearly in the cells as well. I have to say that although I could track the nanocarrier easily I could not track the DNA molecule brought with it so it just might have float into the nucleus somehow.
I haven't seen anything in the literature pointing in one direction or another. I think it's more about personal conception of the system (which is definitely not scientific approach ).
If you don’t have arguments to say that transcription only happens in the nucleus I haven’t seen anything saying that it could happen in the cytoplasm either…
I think it is generally agreed that the transfected DNA has to enter the nucleus and DNA-mediated transcription occurs in the nucleus. mRNA is then transported out of the nucleus for protein synthesis.
The bulk of the transfection complex are present in the cytoplasm and only very small fraction of the DNA enter the nucleus. The level is usually below the detection limit, but it can be detected under right condition.
thanks guys, so eventually it is still transcription in the nucleus but still there is chance it can be happen in the cytoplasm due to all the factors needed can be found in the cytoplasm. right ?
The factors needed for transcription are transcription factors (proteins), polymerase (a protein) and your DNA. As your DNA migrates through the cytoplasm it might encounter some freshly translated transcription factors and polymerase (as said, they are both proteins so they will be transcribed in the nucleus, after which the mRNA gets translated in the rybosomes, located in the cytoplasm/on the endoplasmatic reticulum).
One thing I'm not taking into account are post-tranlational modifications. I'm not an expert (others might shed some light on this), but it might be that your transcription factors and/or polymerase need extra modifications (phosphorylation, methylation) that only happen in the nucleus before becoming active. In that case it wouldn't happen in the cytoplasm.
SO, in theory, it might happen, but I don't believe you would notice.