Transcription: Unit, Machinery & Process of Transcription

Transcription: DNA contains information for the synthesis of specific proteins of the cell. DNA is located in the nucleus of eukaryotes and found in the form of nucleoids in prokaryotes. DNA does not move out from the nucleus to the site of protein synthesis, i.e., ribosomes (located in the cytoplasm), to directly guide the process. Instead, it transfers the information to mRNA molecules which further move to the ribosomes to direct the process of protein synthesis. Hence, an mRNA strand is firstly synthesised over a DNA template. This phenomenon is called transcription. It can be said that the process of transcription involves rewriting the genetic messages coded in DNA into the RNA molecule. The process of transcription follows certain steps in sequence. Let’s read the article to enhance and elaborate on the entire mechanism of transcription in detail.

Fig: Transcription

Transcription Definition

Transcription is the process of synthesising RNA (mRNA) over a DNA strand ( coding strand) with the help of RNA polymerase. Both the strands of DNA do not transcribe RNA; only one out of two strands participate in the transcription. The synthesised RNA further carries the information to encode the protein to the ribosomes located in the cytoplasm.

RNA Polymerase – A Transcriptional Enzyme

RNA polymerase is a multi-unit enzyme that transcribes the RNA over a DNA template and is hence also called DNA dependent RNA polymerase enzyme.

In prokaryotes, only one type of RNA polymerase is found to be involved in the process of transcription that comprises five types of subunits, namely, two alpha subunits and one subunit of each beta prime beta, omega, and sigma. The sigma factor binds to the core RNA polymerase forming the holoenzyme. After the initiation of transcription, the sigma factor dissociates from RNA polymerase. The RNA polymerase further proceeds with the elongation of the RNA chain.

In eukaryotes, three different types of RNA polymerase have been noticed that transcribe RNA. Each comprises about eight or more subunits, involving the four similar to the prokaryotic RNA polymerase. The three different RNA polymerase and their function are listed below:

Transcription Unit

Only one of the two strands is involved in the synthesis of RNA that takes part in transcription. The segment of the DNA that takes part in transcription is called the transcription unit. A transcription unit has three components:

1. A Promoter
2. A Structural Gene
3. A Terminator

The promoter and the terminator bordering a structural gene is a transcription unit. The promoter is located towards the 5’ end (with respect to the polarity of the coding strand). It provides the binding site for RNA polymerase.

A structural gene codes for an RNA or protein product other than a regulatory protein (regulatory factor). It may code for structure proteins, an enzyme, or an RNA molecule. The structural genes may be monocistronic (mostly in eukaryotes) or polycistronic (mostly in prokaryotes). In eukaryotes, the monocistronic structural genes have interrupted coding sequences, i.e., the genes are split or separated. The coding sequence is called the exons, and the interrupted sequence is called the introns. The introns do not appear in processed RNA.

A terminator is a region on a DNA strand that causes the termination of the transcription process. The termination sequence is located at the 3’ end of the coding strand.

Fig: Schematic Representation of Transcription

Machinery of Transcription

The following materials are required for transcription:

1. The enzyme RNA polymerase
2. A DNA template
3. Four types of ribonucleoside triphosphates (ATP, CTP, GTP, and UTP)
4. Divalent metal ions Mg⁺² and Mn⁺² as a cofactor.

Mechanism of Transcription

The process of RNA transcription involves the following steps:

1. Binding of RNA polymerase to DNA duplex: On a signal from the cytoplasm, the histone coat protecting the DNA double helix at the region of the gene to be transcribed is removed, thereby exposing the polynucleotide sequences of the specific region of DNA. The enzyme RNA polymerase binds to a specific site, called a promoter, in a DNA double helix.
In prokaryotes, the RNA polymerase, together with the sigma factor, is called the RNA polymerase holoenzyme.The enzyme recognizes the promoter by its sigma (σ)subunit in prokaryotes and by many transcription factors in eukaryotes. The promoter site is located on the 5’ side and signals where to start RNA synthesis. The promoter also determines which DNA strand is transcribed. Transcription factors and RNA polymerase bind to the promoter together, forming a transcription initiation complex.
Eukaryotes have three types of polymerases, I, II, III, while prokaryotes have only one RNA polymerase. In eukaryotes, the promoter region consists of a specific sequence of DNA called the TATA box to which the RNA polymerase binds and initiates the synthesis of the RNA chain.

2. Exposure of RNA bases: The RNA polymerase moves along the DNA and causes local unwinding and splitting of the DNA duplex into two chains in the region of the gene to be transcribed. This exposes the A, T, C, and G bases that project into karyoplasm from the phosphate- deoxyribose sugar backbone. Only one strand, called the sense strand of DNA, functions as a template. The other strand is complementary. It is called antisense, or a non-coding strand.

3. Base-Pairing: The ribonucleoside triphosphates (ATP, GTP, CTP, UTP) that floatly in the karyoplasm of the nucleus serve as the raw material for RNA synthesis. They are formed by the activation (phosphorylation) of ribonucleoside monophosphate (i.e., AMP, GMP, CMP, UMP) through union with ATP. The enzyme phosphorylase catalyses the activation process.

4. The three steps comprise the initiation of the RNA chain.Dissociation of sigma factor: After the initiation of RNA chain formation, the sigma factor dissociates from the core enzyme to carry out the elongation of the RNA chain.

Fig: Base-pairing between DNA and mRNA.

5. Formation of RNA chain: With the energy so released, each ribonucleoside monophosphate joined to the DNA template chain further joins the ribonucleotide that has to arrive earlier and thus making the RNA chain longer. This process is called the elongation of the RNA chain. The enzyme RNA polymerase catalyses the formation of the RNA chain along with the Mg⁺² and Mn⁺² ions.

6. Separation of RNA chain: The enzyme RNA polymerase, when reaches and identifies the terminal signal on the DNA template, the RNA chain is released. This has been observed in most of the eukaryotes.
However, in some prokaryotes such as E.coli, a specific chain termination protein called rho factor terminates the synthesis of the RNA chain.

7. Winding of DNA segment: As the RNA chain grows, the transcribed region of the DNA coding strand twists with the non-sense strand of the DNA through hydrogen bond, and the two strands thus coiled with each other, and the RNA polymerase and RNA chain are completely released. Further, the histone protein coat is added again to the DNA duplex.
The separation of the RNA chain and the winding of the DNA segment causes the termination of transcription.

Fig: Mechanism of transcription

Post Transcriptional Changes: Processing of RNA

RNAs transcribed originally from the DNA are called primary transcripts that undergo several changes before they become functional in eukaryotes and prokaryotes.

Transcription in eukaryotes is comparatively more complex than in prokaryotes. In eukaryotes, the transcriptional unit has only one gene. The newly formed RNA synthesised by RNA polymerase II is called hnRNA or primary transcript that contains both unwanted or non-sense base sequences (introns) alternated with useful base sequences (exons). The hnRNA is modified into functional mRNA involving the following three steps:

I. Capping at 5’ end: A cap containing unusual nucleotide (methyl guanosine triphosphate) is added at the 5’end of hnRNA. The methylated cap, besides providing a ribosome binding site, protects the hnRNA from degradation by nucleases.

II. Tailing at 3’end: It is called polyadenylation. It involves the addition of a Poly-A tail at 3’end with the help of an enzyme poly-A polymerase. The sequence AAUAAA (10–30 nucleotides upstream of the polyadenylation site) is important for cleavage and further causes the polyadenylation of mRNA.  Polyadenylation might protect the 3’end from the degradation by exonucleases.

III. Splicing: It is the process of removal of introns (non-coding segments) through cutting and joining the exons together. The process is carried out with the help of snRNPs (small nuclear ribonucleoproteins). The latter is formed by the association of proteins with nuclear RNAs. The ends of the adjacent exons are joined together by a ligase enzyme.

Fig: RNA Processing

The completely processed hnRNA is now called mRNA that is released from the nucleus to the cytoplasm for the synthesis of proteins. It can be said that transcription is a mechanism by which the base sequence of a DNA sense strand is copied into a complementary base sequence of RNA. Thus the RNA synthesised on the DNA template is complementary to the sense strand and is identical to the non-sense strand, except that thymine is replaced by uracil.

Summary

Transcription is the process of synthesis of RNA over the template strand of DNA. The process is initiated with the unwinding of DNA strands and is catalysed by the RNA polymerase enzyme. The sigma factors of the RNA polymerase enzyme recognises the promoter site on DNA and initiate the process of transcription. The synthesis of the RNA chain continues till the recognition of terminal signals or rho factors (in E.coli) that are responsible for stopping the synthesis of RNA. The synthesised RNA undergoes further processing to become functional in prokaryotes and eukaryotes. DNA stores the information for protein synthesis but does not leave the nucleus. Hence transcription is essential to further proceed with the translation of genetic code in the form of a polypeptide chain on the ribosome located in the cytoplasm.

Frequently Asked Questions on Transcription

Q.1. What are the three main steps of transcription?
Ans: The three main steps of transcription are initiation, elongation, and termination.

Q.2. Which enzymes are involved in the initiation of eukaryotic transcription?
Ans: The transcription of RNA in eukaryotes involves the three polymerase enzymes, i.e., RNA polymerase I, RNA polymerase II, and RNA polymerase III.

Q.3. What is the role of sigma factors RNA polymerase?
Ans: Sigma factor is responsible for the recognition of promoter genes on a DNA template.

Q.4. What is the site of mRNA transcription?
Ans: The site of mRNA transcription is the nucleus of the cell.

Q.5. What happens during RNA splicing?
Ans: RNA splicing is the process of removing introns (non-coding segment) that are found in between the exons (coding segment) and further joining the exons in a sequence with the help of ligase enzyme.