In this article we will discuss about the mechanism of eukaryotic gene expression, particularly in humans.
1. In multicellular organism, each cell expresses a subset of its genes.
2. A cell expresses different genes depending on its growth state or environment.
3. Sometimes gene transcripts are spliced differently in different cells.
4. Each gene carries not only structural information but also instructions for its correct expression in space and time.
5. As in case of prokaryotes (promoter), eukaryotic genes also found to share common motif in their 5′-flanking region.
6. One of the important motif was a sequence TATAA, often found about 25 to 30 bp 5′ to the start of transcription (this sequence resembled the -10 element in bacterial promoter).
7. Other sequences commonly found upstream of genes include CCAAT and GGGCG (GC boxes).
8. The position of CCAAT and GC box were variable but they were always farther from the -gene than was TATAA.
9. All of these three sequences control the eukaryotic transcription.
10. Enhancers are the other sequence found in cellular genes which are generally 50-150 bp long.
11. Unlike bacterial promoter elements which is generally constituted a single short sequence motif enhancers are more complex and in enhancer there are redundant sub-elements.
12. Enhancers are tissue specific and/or regulated by signals.
13. The primary function of the enhancer is to bind cellular transcription factors.
14. There are generally two broad kinds of protein factors of which some are known as general factors required for initiation at all promoters while others are gene-specific and required only for certain promoters.
15. Of the different general factors, transcription factor II D (TF IID) which binds to the TATAA sequence.
16. Actually the gene specific transcription factors are DNA birding proteins.
17. Enhancers have the property to activate gene expression over long distances.
18. Transcription factors are found to contain two functional domain, one for DNA binding and other for transcriptional activation.
19. The DNA binding domain of the transcription factors fall into several structural families which are basically based on their primary amino acid sequence.
20. Transcription factors are classified into 5 major categories:
(a) Homeodomain protein:
These are quite similar to prokaryotic DNA binding protein.
(b) Zinc finger protein:
Have repeated motifs of cysteine and histidine and its three-dimensional structure is coordinate by Zinc ion. e.g. TFIIIA a factor for RNA Polymerase III.
(c) Leucine Zipper protein:
Have 4-5 leucine residues repeats forming a dimer in zipper-like fashion.
(d) Helix-Ioop-helix (HLH) Protein:
Structurally these are very similar to that of leucine zipper but here is no leucine repeats,
(e) Winged helix (Forkhead) Protein:
DNA binding domain in H5 (histone).
21. Transcription factors are modular i.e. the activation domain of one factor can be joined to the DNA binding domain of another and the resulting hybrid protein is fully active in cells (this cross-binding may be due to the cAMP regulated stimulation).
22. In Eukaryotes there are 3 kinds of RNA Polymerases which are:
(a) RNA Polymerase I: transcription of rRNA.
(b) RNA Polymerase II: transcription of mRNA.
(c) RNA Polymerase III: transcription of tRNA and other small nuclear and cytoplasmic RNA.
23. There are different transcription factors for different RNA Polymerase and also different types of promoters for control of transcription. In case of RNA Polymerase I and II, transcription is regulated by some sequences (like TATAA, GC box, CCAAT box etc.) which are generally upstream or downstream of the gene concerned, wile in case of RNA Polymerase III transcription is regulated by sequences in the middle of the gene.
Following are list of some transcription factors:
24. As the organization of eukaryotic genome i.e. the chromosomal DNA is tightly packed into higher-order nucleoprotein structures, the basic unit of which is nucleosome (a complex of eight histone protein around which 200 bp of DNA exist). Transcription factors might act to free DNA from nucleosome so that promoter is accessible to the large transcription complex.
25. It is now accepted that rRNA has an important functional role during the translation of mRNA and there is base paired interactions between mRNA and rRNA during initiation, elongation and termination of protein chain.
At the time of initiation the relative level of translation of different mRNA is determined in part by the complementary between the shine- Dalgamo sequence of the mRNA (AGGAGGU at 5′ end) and the anti-shine- Dalgamo sequence near the 3′ end of 16s rRNA (UCCUCCA at 3′ end of 16s DNA).
26. Many other factors also influence the seletion of specific mRNA for translation, which are Shine-Dalgamo sequence and an initiation Codon (AUG or GUG), a suitable spacing between the two (SD sequence and initiation codon), a translational enhancers sequence (e.g. Epsilon sequence, UUAACUUUA; McCarthy’s enhancer UUAAUUUAC etc.).
27. Like the transcription factors mentioned above, the initiation phase of protein synthesis in eukaryotic cells is promoted by at least 10 different initiation factor proteins. The rates of translation are controlled by regulating initiation factor activities of the different initiation factors the IF-2α, β, ϒ and IF-4. A, B, D and E are most important and their functions during initiation as follows:
IF-4A-ATP binding protein with RNA dependent ATPase activity
IF-4B- helps mRNA binding to ribosomes and appears to promote the functions of IF-4A, F.
IF-4D- acts late in the initiation pathway, following 80s initiation complex formation, by promoting the formation of first polypeptide bond.
IF-4E- binds to the 7-methyl-guanosine cap structure of mRNA and is a component of the cap binding complex, IF-4F.
28. Not only the initiation factors (IF) but also the elongation factors (EF), ribosomal protein s6 and their change by phosphorylation state may alter the rates of translation process.
29. Human genes have complex sets of cis- acting transcriptional control sequences and the transcription factors are transacting elements which recognise and bind specific cis-acting sequence elements. Human genes show 4 or more classes of cis acting regulatory elements.
Each class is typically made up of multiple short sequence elements distributed over a few hundred base pairs. However, the different classes which regulate a single gene may be located at considerable distances.
Following are the recognized classes of cis-acting element for individual gene:
(a) Promoters:
These are located in the intermediate upstream region of the gene (generally within 200 bp from the transcription start site) and serve to initiate transcription e.g. CCAAT box, TATAA box and GC box.
(b) Response elements (REs):
These are found only in selected genes whose expression is controlled by an external factor, such as hormone, growth factor or by an internal signalling molecule cAMP. These are located a short distance upstream of the promoter element (generally within 1 kb of the transcription start site).
(c) Enhancers:
These are positive regulatory elements whose functions, unlike those of promoters, are independent of both their orientation and, to some extent, they regulate their distance from the genes. They often contain within a span of 200-300 bp and are recognized by tissue-specific transcription factors.
(d) Silencers:
These are the negative regulatory elements and very close to the promoters, both upstream and also within the introns.