Monthly Archives: September 2018

It’s tempting to think of genes as simply a series of nucleotides beginning with a START codon and ending with one of the three STOP codons. However, there are a number of additional regulatory elements that must be present in order for a gene to be transcribed and translated appropriately.

Transcription is regulated by signals for the DNA-dependent RNA Polymerase ( or simply ‘RNA Polymerase’) to attach and detatch from the DNA in the nucleus.

The attachment point is known as the promoter, and ‘Initiation’ of transcription is characterized by the recruitment of the RNA polymerase to the DNA upstream of the coding sequence. Polymerase engagement unwinds the DNA allowing for recruitment of ribonucleotides and the start of RNA synthesis (Elongation). There may be a number of false starts until a sufficiently long RNA is made to stabilize the enzyme, and even after elongation begins, it may stall and restart until the polymerase reaches a termination signal. There are a number of different kinds of termination signals, but they all occur downstream of the stop codon and serve to disengage the polymerase from the DNA (Termination) so that it is free to recycle back to the promoter.

There are some key differences between the ways that prokaryotes and eukaryotes perform these operations, but all the above elements occur in each system. The key to understanding this clearly is that transcription must occur before translation, and therefore, the transcribed region must have all the translated region within it. This sounds obvious, but can be helpful in order to envision the gene correctly as a physical object.

Imagine that you are a scientist interested in cloning gene A. You’ve just amplified the entire gene A including some flanking sequence by PCR and run the resulting amplicon onto a 2% gel with a 100 bp ladder. See the results in Figure 1.

Happy with your result, you clone the DNA into a cloning vector, pCR2.1 where you can make up tons of DNA to work with. These cloning vectors are great for making lots of DNA, but they do not express any of the genes as proteins (i.e. the DNA is replicated, but not transcribed and translated.) Because you do want to express protein, you need to subclone your gene from the cloning vector into an expression vector. To complicate matters, the gene needs to go into the expression vector with the promoter upstream of the gene and the poly A signal downstream of the gene (See Figure 2). The promoter is the location that the RNA polymerase binds to transcribe the gene, the polyA site is what signals the polymerase to add a polyA tail to the mRNA.

In order to clone your gene into the expression vector, you decide to determine the direction that your gene has inserted into pCR2.1. To do this, you take advantage of the fact that there is a NotI site off-center in the insert and also one in the plasmid (See Figure 3). Gene A is just under 900bp long in total, the Not I site is located at position 800bp.

You cut the plasmid with NotI expecting either an ~800bp band or a ~100bp band depending upon the orientation of the insert. The results of your digest are seen in figure 4, leading you to believe that your insert is in the direction seen in figure 5.

In order to subclone from the cloning vector into the expression vector (figure 6), you cut the gene out of pCR2.1 with SpeI and NsiI and isolate the ~900 bp fragment. The same two enzymes can be used to open the expression vector and isolate the linear plasmid. The two fragments can then be combined in the presence of DNA ligase to complete the subcloning.