Goals
- Describe the major differences (polymerases, mechanism of initiation, promoter, termination) between eukaryotic and prokaryotic transcription
- Describe the structure and function of the 5′ cap and polyA tail of an mRNA
Transcription Overview
An enzyme called RNA polymerase is used on a template DNA to create a new strand of RNA. The key steps to this process is initiation, elongation, and termination.
There are different types of RNA polymerases which create different products, see the table below.
| RNA Polymerase | Products |
| Prokaryotic RNAP | All RNA |
| Pol I | rRNA |
| Pol II | mRNA, miRNA |
| Pol III | tRNA |
RNA Polymerase is a highly conserved molecule. In E. coli with a 5 protein subunits. It has two α (alpha) subunits αI and αII two β (beta) subunits, an ω (omega), and finally a σ (sigma) subunit. However, in eukaryotic cells the three different polymerases all have the beta, alpha, and omega subunits, but differ in the sigma subunit as eukaryotic have common subunits.
Like previously mentioned, in prokaryotes RNA Polymerase is enough to initiate transcription. In eukaryotic cells on the other hand, accessory proteins are required to initiate transcription. These accessory proteins are know as General Transcription Factors (or GTF’s)
GTF’s are required for initiation of transcription in eukaryotes, but note that there are also specific transcription factors which regulate gene expression at specific promoter regions, or genes.
TFIID which is short for Transcription Factor Pol II D which is required for initiation in Eukaryotes, a protein associated with this transcription factor known as the TATA Binding Protein (TBP) binds to a region known as the TATA box (consists of a polyTATA sequence, also known as the promoter sequence). The TFIID and TBP, once bound, recruit RNA polymerase II alongside TFIIH (which contains helicase activity), and initiation has been achieved.
Prokaryotic initiation only contains 6 polypeptides, whereas Eukaryotic initiation contains a much more significant amount (greater than) > 30 polypeptides.
In gist, DNA is bound to, the promoter site is recognized, TFIIH melts the DNA with its powerful helicase activity, and voilà you’ve got transcription started.
TFIIB is bound at the recognition element upstream of the initiator at -37 to -32, the TATA box is recognized upstream at -31 to -26 of the Initiator (-2 to +4), which is bound to by TFIID, another TFIID is also bound downstream at +28 to +32 at the Downstream promoter element (DPE).
The promoter regions is located at -35 and -10 downstream of the initiator.
After the initiator has officially commenced transcription, RNA polymerase continues with elongation, an active process where TFIIH phosphorylates RNA Pol II. RNA Pol II has elongation factors (capping, splicing, and polyadenylation).
These elongation factors will be discussed in another post, but to summarize. The capping factor immediately adds the 5′ cap, once the termination sequence is met (AAUAAA), an endonuclease event occurs removing the pre-mRNA.
The pre-mRNA is immediately given a polyA sequence at ~100-250 base pairs in length to prevent degradation. Known as the 3′ polyA tail.
RNA polymerase continues to process mRNA, however it is not protected, and a protein degrades the new mRNA being produced in an exonuclease event and disrupts the RNA:DNA helix effectively terminating transcription.
The 5′ and 3′ ends of the pre-mRNA are also bound to cap specific proteins, such as translational machinery.
Credit: https://www.youtube.com/channel/UC8wZzNSXUHzv3QijdOzeELA