0.1 Genetics background  (Page 3/3)

Transcription is the process of creating RNA from DNA. Transcription is also the point at which most of the regulation of gene expression occurs and because of this it is a very complex process, especially with regard to its initiation. To say that DNA is transcribed to RNA is a nice (over)simplification, but we need to delve a little deeper into the details to really appreciate what is going on during transcription. A more complete view of transcription includes five steps: 1) transcription of DNA to pre-mRNA, 2) a 7-methyl guanosine cap is added to the 5' end of the transcript, 3) a poly(A) tail is added to the 3' end of the transcript, 4) the introns are spliced out of the pre-mRNA, which finally yields, 5) the mRNA transcript proper.

Because the first step, the initial transcription of DNA to pre-mRNA, is the most involved, I am going to hold off on discussing it for a moment and expand on steps 2-5 first. (2) The addition of the 5' 7-MG cap is important for two reasons: the 5' caps are recognized by protein factors that initiate translation, and it also helps protect the transcript from nucleases. Nucleases are very common in the cell and because of this unprotected RNA has a very short half-life inside the cell. Nucleases are actually so common that working with RNA in the laboratory can be quite difficult because the samples have a tendency to disintegrate into useless bits. (3) The poly(A) tails are formed in a two step process: an endonulcease cleaves around 1000-2000 non-coding bases from the 3' end of the pre-mRNA transcript and then poly(A) polymerase adds 20-200 AMP molecules to the 3' end of the transcript. The poly(A) tail is important in the cellular transport of the mRNA transcript and, like the 5' cap, also helps to stabilize the mRNA transcript.

Once the 5' cap and the poly(A) tail have been added, only one step remains for the pre-mRNA transcript to be complete and graduate to mRNA status: splicing. Eukaryotic genes contain two types of transcribed regions: introns and exons. Exons are the regions of the genome that contain actual coding information. Introns are non-coding, meaning that intronic sequences are never translated to protein, in fact they are never included in the final processed mRNA transcript. Splicing is the process of removing introns from the pre-mRNA transcript to produce an exon-only mRNA molecule, which is then shipped off for translation. Generally, eukaryotic mRNAs are considered to monogenic. However, up to one fourth of the transcripts in C. elegans have been show to be multi-genic (i.e. they contain exons from multiple genes).

A further complication of the splicing process is that mRNA can undergo alternative splicing. To illustrate this let's imagine a gene that has 3 exons and two introns. From this gene, three different final transcripts are possible. In all transcripts the two introns are going to be removed, however, the cell can combine the exons however it wants as long as the original order is maintained. This means that for this example the possible mRNA transcripts include: Exon1-Exon2, Exon1-Exon3, and Exon1-Exon2-Exon3; however, Exon3-Exon1 is not possible because the exons are out of order.

An interesting side note is that some introns are capable of self-splicing, that is they can politely remove themselves without the intervention of any proteins. This is significant mainly because it is a significant counter example to the idea that RNA is an inert transcript and action is soley the domain of proteins. RNAs should really be viewed as having both enzymatic properties and abstract information-carrying ability. Because of this many people believe that RNA was the original genetic molecule and that DNA and proteins evolved later in the game.

Alternative splicing is a very important and powerful tool. To understand the benefit alternative splicing gives the cell we need to understand something about proteins. Proteins can be understood as containing modularized functional units. These functional units can be active sites on enzymes, large structural motifs such as beta-sheets or alpha-helices, or motifs that direct the eventual destination of expressed proteins. A good example of an alternatively spliced pre-mRNA transcript is the mouse IgM immuoglobulin transcript. IgM exists in two forms: excreted and membrane bound. These two forms of the protein differ in the only in the C-terminus: the secreted protein has a secreted terminus motif while the membrane-bound protein has a C-terminal membrane anchor region. Both products come from the same pre-mRNA, but alternative splicing includes either the terminal exon that creates the excreted form of IgM or the membrane-bound form of IgM.

This is a good time to take a step back from our discussion, take a deep breath, and summarize what we have covered so far. (1) DNA exists as a double stranded helix that is both complimentary and antiparrallel. (2) DNA in vivo exists in a very compact and regular structure of nucleosomes, 30nm fibers of braided nucleosomes, and loops of fibers. (3) The central dogma of genetics: DNA is transcribed to RNA, which is then translated to proteins. (4) DNA is the stable, long-term form of genetic information. (5) RNA is (mostly) an intermediary between DNA and the protein-making-factories, ribosomes. (6) RNA transcription is not nearly as simple as the central dogma might lead you to believe. Which leads us to the point I put off earlier: how is transcription initiated in the eukaryotic genome?