Eukaryotic mRNA consists of several parts: the 5′ Cap, 5′ UTR, coding sequence, 3′ UTR, and 3′ poly(A) tail. The poly(A) tail plays a critical role in stabilizing and enhancing the expression efficiency of mRNA. When mRNA is produced through in vitro transcription (IVT) using a plasmid template, the poly(A) tail is often encoded in the plasmid template for ease of the transcription process.
The conventional process to produce more plasmids from the starting plasmids is to use E. coli fermentation. In the fermentation process, E. coli cells take up the starting plasmids and use the material from the cells to produce more copies of the plasmids. However, one observed issue is that most strains of E. coli do not preferentially transcribe the poly(A) tail of the plasmids, making it difficult to maintain the original length of the poly(A) tail. Sometimes, after fermentation, the poly(A) tail can decrease from 100 to 20 adenine (A) residues. Without a sufficient length of the poly(A) tail from the plasmid template, it would not be possible to produce high-quality mRNA.
So, why doesn’t E. coli like to transcribe the poly(A) tail?
The main difference lies in the variations in the structure and processing of mRNA between prokaryotes (like E. coli) and eukaryotes (like humans). More specifically, the reasons are as follows:
- Lack of a Nucleus: One key difference between prokaryotes and eukaryotes is that the former lack a nucleus in the cell. After pre-mRNA is transcribed, it needs to go through multiple post-transcriptional processes to mature, including capping and adding a poly(A) tail. These post-transcriptional modifications happen in the nucleus of eukaryotes, and E. coli, being a prokaryote with no nucleus, does not possess the machinery for these processes.
- Different mRNA Structure: Eukaryotic mRNA contains a 5′ cap and a 3′ poly(A) tail as part of its mature structure. In contrast, in prokaryotes like E. coli, both structures are missing. Instead, there’s a Shine-Dalgarno sequence near the 5′ UTR, which is important for ribosome recognition and binding, and a terminal sequence at the 3′ end. Therefore, most strains of E. coli are not accustomed to the different structure of eukaryotic mRNA.
- Transcription and Translation Coupling: In eukaryotes, transcription and translation occur at different times and in different cellular compartments. mRNA is transcribed and modified first in the nucleus, and then it is translocated into the cytoplasm for translation into protein with the help of ribosomes. In prokaryotes like E. coli, these two steps are coupled, and ribosomes begin translating even before the transcription process is complete. Therefore, no post-transcriptional modifications are needed, such as adding the cap and poly(A) tail.
Overall, most strains of E. coli are not adapted to the structure of eukaryotic mRNA, do not share the same transcription-translation process, and lack the nucleus required for post-transcriptional modifications. Therefore, it is common for E. coli fermentation to result in the loss of part or all of the poly(A) tail after transformation. However, there are some engineered strains that are able to express eukaryotic genes, including the poly(A) tail. Nevertheless, in their natural state, bacteria like E. coli have evolved distinct mechanisms for mRNA processing and translation that differ from those found in eukaryotes.