Intricacies of Eukaryotic Transcription

The transcription process in eukaryotes involves a very complex process. It involves the synthesis of DNA followed by transcription. The complexity of the transcription process makes it important for us in knowing every detail. Studying transcription helps us in knowing more about the functionality of the molecules and processes known as protein synthesis. Unlike prokaryotic RNA polymerases, the eukaryotic polymerases involve three classes. After RNA synthesis, the molecule gets modified further. Hence, it is important to know the E. coli transcription process to clarify and study the same process in eukaryotes. Although similar to prokaryotes, eukaryotic transcription is different.

Eukaryotic RNA polymerases:

RNA polymerase-I is known to catalyze the synthesis of ribosomal RNA such as 28S, 18S, and 5.8S rRNA. RNA polymerase-II is known to synthesize messenger RNA (mRNA) and some small nuclear RNAs (snRNA). RNA polymerase-III synthesize transfer RNA (tRNA), 5SrRNA, and snRNA not synthesized by other polymerases. 

RNA synthesis:

The process of RNA synthesis is very complicated. The molecules involved in the process such as RNA polymerases also exhibit a complex nature. They have multiple subunits and encoded by several genes. There is very little information about these enzymes. Mainly RNA polymerase-II involves in transcribing the genes. The transcription process produces a precursor mRNA. It is modified to produce a mature and functional mRNA. Like prokaryotic cells, eukaryotic cells also consist of promoters. The promoter functions in eukaryotes involve more sophisticated technique as compared to prokaryotes. There are two parts of the promoter elements such as core promoters and promoter-proximal elements. The core promoters are known as cis-acting elements. These elements at 50 base pair upstream work well at or near the transcription site. They are necessary for starting the transcription process at the right site. Core promoter elements are short sequences or initiator (Inr) elements and TATA box elements. The Inr elements span the transcription site. The TATA box elements bind to RNA polymerase via TATA-binding proteins. These elements are analogous to Pribnow box in prokaryotes. The TATA box or Goldberg Hogness box consists of a seven nucleotide consensus sequence known as TATAAAA.

The core promoter also consists of general transcription factors (GTFs) required for initiation of transcription. The GTF labeling is specified based on the function. There are six types of GTFs such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. TFIID binds to TBP of TATA box to form a complex. It is a binding site for other transcription factors such as TFIIA and TFIIB. Later on, TFIIF and RNA polymerase II bind and form a minimal transcription initiation complex. Later on, TFIIE and TFIIH bind to form complete transcription initiation complex. 

Core promoter	Normal function	Effect of mutation
Inr elements	Direct the initiation of transcription	Any mutation in the Inr elements makes the core promoter non-functional.
TATA Box	Helps in the binding of transcription factors and RNA polymerases to initiate transcription.	RNA polymerase cannot bind due to mutation. As a result, transcription does not occur.

Table 1: Promoters and their functions along with their effects after mutation. 

The promoter-proximal elements are present towards or near the place of attachment. In other words, they are upstream of the TATA box. There are two types of promoter-proximal elements such as CAAT box and GC box. The CAAT box consists of CAAT consensus sequence. The GC box consists of a consensus sequence GGGCGG. Both the promoter elements work in synergy. Mutation in these elements significantly affects the rate of transcription. A promoter function depends on both the core promoters and promoter-proximal elements. These elements control the time and way of gene expression. Transcription regulatory proteins help the promoter proximal elements to control the time and way of gene expression. These regulatory proteins are known as activators. The process of transcription becomes inefficient without the regulatory proteins. The promoter-proximal elements become incapable of regulating the transcription without activators.

Maximum efficiency in gene transcription involves another class of sequences known as enhancers. They are also known as cis-acting upstream or downstream elements. A peculiar specialty of an enhancer involves its maximum efficiency of transcription even from a distance. Many enhancers also determine spatial patterns of gene expression in higher eukaryotes.

 

    

Image 1: Initiation of transcription: It includes four main steps. (1) The initial commitment complex forms. The TATA box gets recruited along with the other proteins. (2) After the recruitment of TATA box and TBP, the other two transcription factors such as TFIIA and TFIIB join and initiate the transcription. (3) RNA polymerase-II along with TFIIH join the complex. (4) TFIIE and TFIIF bind and allow the completion of the transcription.

Initiation:

The transcription factors such as TFIID bind to TATA-binding protein on the TATA box. All other transcription factors also bind to this region along with the RNA polymerase II and form a pre-initiation complex. The RNA polymerase II starts synthesizing mRNA using only one strand of DNA double helix. The transcription begins at 5’ end and ends at 3’ end. Appropriate ribonucleotides get added to mRNA due to the activity of RNA polymerase II. The mRNA is a single-stranded molecule. All the sequences of structural genes including introns and exons get transcribed in mRNA. The mRNA is known as primary mRNA because it consists of introns that do not code for any protein. Hence they have the least importance in a functional mRNA. A process known as splicing removes the non-coding regions to give a fully functional RNA molecule.

Processing of pre-mRNA involves capping:

Immediately after synthesizing pre-mRNA, the process of capping occurs. Capping occurs after the RNA polymerase escapes the promoter. Hence, we must know two main processes such as promoter clearance and promoter escape. Promoter clearance involves moving ahead of the promoter sequences to begin RNA synthesis. Promoter escape means polymerase has completed its activity at the promoter site and now moves away from that region to proceed to cap. An extra guanosine group gets added to the extreme 5’ end of the RNA due to the activity of guanylyltransferase. The G- terminal gets methylated at the seventh nitrogen atom due to the activity of guanine methyl transferase. These two enzymes attach to the C-terminal domain of RNA polymerase II. 

Image 2: Steps involved in RNA synthesis and processing in eukaryotes.

Polyadenylation:

The process of polyadenylation accompanies the addition of the poly A tail to the 3’end. The tail is essential for exporting mRNA from the nucleus to the cytoplasm. It also protects it from exonuclease and stabilizes mRNA. Addition of a poly A tail to the 3’ end of RNA is a very complicated process in mammals. There is a poly A consensus sequence on mRNA known as AAUAAA sequence. CPSF also was known as Cleavage and Polyadenylation Specific factor and two other cleavage factor protein bind to and cleave the RNA. Then the enzyme poly-A polymerase adds a poly A tail to the 3’ end.

Elongation of eukaryotic mRNA:

Mammalian cells consist of 13 different elongation factors. The process of elongation starts when the RNA polymerase II has completed the promoter clearance or initiation process. The elongation factors exhibit various functions. The TFIIF, CSB, ELL, and Elongin are the factors suppressing the pausing of the RNA polymerase II. The pausing and stopping affect the activity of polymerases. Pausing arises due to the presence of a hairpin loop or intra-strand base pairs. Hence, these factors help the RNA polymerase II to skip such structures and move ahead. The TFII factor protects from the complete cessation of elongation. It is a chromatin modifier since it interacts with H2A and H2B.

mRNA with poly(A) tail	mRNA without Poly A tail
Helps in the termination process	Have two types of cleavage signals A hairpin loop and a nine nucleotide consensus CAAGAAAGA

Table 2: Types of mRNA based on polyadenylation

Polyadenylation helps in terminating the process of transcription.

Termination

The termination of transcription is favored by changes in the CPSF-CTD interactions. The exact information on the mechanism of termination is not known.

The mRNA consisting of both protein-coding and non-coding region undergoes a process known as splicing. It is a post-transcription process that involves the removal of introns and ligation of exons to form a mature mRNA ready to depart from the nucleus.

References:
[1] Eukaryotic Transcription Factors, David Latchman, David S. Latchman

[2] Molecular Biology of the Cell, Bruce Alberts
[3] IGenetics, Peter Russell
[4] Gene Regulation: A Eukaryotic Perspective, David S. Latchman

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