Translation of mRNA?

Question

We inserted an insulin gene from human into bacteria. Will translation of the gene (protein formation) occur in bacteria? If translation occurs then why does it occurs, give a reason for this?

Answer 1

Yes, bacteria will produce human (or any organism's) proteins if you introduce their genetic material but there are a few things to consider.

First, the introns must be removed from the human genetic sequence. Bacteria do not have the machinery to splice out introns after transcription. This is typically done by using a viral protein to reverse transcribe mRNA, which already have the introns removed, into DNA which you can use to transform bacteria.

Second, you need to insert a Shine-Dalgarno sequence. This allows the bacterial ribosomes to bind to the mRNA they will produce, and translate the protein you desire. This sequence is not found in eukaryotic genes, so you have to make sure it was included either in the vector or in the PCR primers you used to amplify the gene sequence.

Answer 2

Although the genetic code is almost conserved among organisms there are some issues to take in account, among which:

• Every organism has its own codon usage, i.e. the differences in frequency of occurrence in synonymous codons in coding DNA (that reflect the composition of tRNA abundances). So, for example, the codons UUU and UUC both encode for phenylalanine but - I'm making this up - UUU is common in human but rare in coli while UUC the contrary. If the coding sequence of insuline is rich of UUU, you probably need to change the codon UUU to UUC in order to avoid slow translation.
• Also the aminoacid abundance can be very variable between two organisms. One of the ways you can handle the differences is by "tuning" the expression of the aminoacids. Here a table of aminoacids abundance in coli.
• The regulatory sequences in coli are not the same as in human. In fact, prokaryotic translation initiation needs a purine-rich sequence upstream the (usually) AUG initiation codon called Shine Dalgarno. This sequence is complementary to the 16S RNA in 30S ribosome subunit.
• Often peptide chains are subjected to various post-translational modifications. The insulin peptide, for example, once synthetized is cleaved by specific peptidases and disulfide bonds are formed. Because human and coli post-translational modification mechanisms are not the same, this can be a difficult problem to handle. Instead of trying to exactly reproduce the protein with all its modifications, often people search for functionally (and not structurally) identical proteins.
• Also the folding process can be different, both assisted (by chaperones) and spontaneous (withouth chaperones). The folding can be influenced by a number of factors like molecular crowding, pH, ...
• A lot of eukaryotic genes have introns: sequences removed before translation. Once splicing sites are known, this problem is easily solved by using a sequence withouth introns.

Answer 3

All the organisms share a common ancestor and have evolved from it. So they share quite the same machinery for transcription and translation. Codons are also interpreted in the same way in almost all the organisms.

So, yes translation should happen ( Assuming that the gene has been cloned in the right way.)

Answer 4

It works, and is actually done to make all the "human" insulin which is used by diabetes patients today. This is because the genetic code is almost completely conserved (with only very few exceptions in nature), so bacteria use the same codons to code for an amino acid as in humans. So with a suitable vector (a ring of DNA which carries extrachromosomal genetic information) which has the ability to replicate independent from the cell and which has the correct promoters (start points) for initiating transcription in bacterial cells, you can basically clone and express any human gene.