Submitted by kathmkath t3_zgxhqw in askscience
If a plasmid has five ORFs to code for five different proteins, does it all get transcribed to a single rna transcript which then gets translated to five different proteins due to start stop codon? OR does each ORF get transcribed to a single rna transcribed which leads to an individual protein? What determines the start stop signal for rna transcription?
sometimesgoodadvice t1_izkdgst wrote
Much like there are defined translation start and stop sites (ATG and TAG/TAA/TGA, respectively), there are translational and start and stop sites. These tend to be more complicated as there is variability in the UTRs of the RNA. The mechanisms are different in prokaryotes and eukaryotes and plenty of exceptions exist.
Without going into too much detail, there are different protein that interact with DNA and also with elements of the RNA polymerase complex that will start initiation. These are known as transcription factors and different ones will bind different DNA sequences. This is how you have control over which genes are expressed when. If there is a presence of a transcription factor, it will help initiate transcription of those genes whose upstream sequences it binds to. The whole region where this is occurring is known as a "promoter".
Similarly, there are sites where transcription halts. In translation, the stop codon works as a stop site because the tRNA that are complementary to the stop codons are not charged with amino acids. So they cannot add an amino acid to the chain and subsequently cant come off and create a new site for addition of more amino acids. There is no energy release from the peptide bond formation, so the ribosome does not move forward on the mRNA and simply sits there. Eventually (this is pretty fast) the ribosome dissociates and the mRNA and protein go their separate ways. Transcriptional stop sites work very analogously and are called "terminators". However, since there is no "empty" nucleotide to attach to the RNA, instead terminators use structure to simply stop the RNA pol from moving forward. This is usually done through the single stranded DNA sequence being such that it natively forms tight hairpins which the helicase cannot unwind. The polymerase falls off, and then eventually the double stranded DNA is zipped back up.
For the practical question of your answer. When designing a plasmid, you can have multiple promoters and multiple terminators. Bacteria can translate multiple proteins from the same rna, eukaryotes generally can't so they will require a promoter-terminator pair for every ORF. There are certain techniques around that, but that's beyond the scope here. In general, you want to avoid repeating the same promoter or terminator in a plasmid since that can cause recombination and the transcription factors will be divided up between the different promoters causing variability in expression levels between the cells. Whether you put multiple proteins on the same transcript or different one in bacteria depends on your goal. Proteins on the same transcript will be expressed roughly in the same ratio, since the amount of mRNA for one protein is exactly the same as the amount for the other. If you want differential control over the expression of one vs another protein, then they will be under action of different promoters.