Transcription

Transcription

Transcription is the biological process through which ribonucleic acid (RNA) is synthesized using DNA as a template. It marks the first step in gene expression, where the genetic information stored in DNA is transferred to RNA.

                                   

In this process, the sequence of nucleotides in DNA is copied into a complementary strand of RNA. Among the various types of RNA, messenger RNA (mRNA) plays a crucial role. It carries the genetic code from the nucleus (where DNA resides) to the cytoplasm, where proteins are synthesized by ribosomes. Hence, mRNA acts as a messenger between DNA and the protein-synthesizing machinery.

During transcription, only one of the two strands of the DNA double helix serves as the template strand, also known as the non-coding or sense strand (Note: Some textbooks may switch these terms, so always refer to specific usage). This strand provides the pattern for the sequence of nucleotides in the RNA molecule.

The other DNA strand, which does not participate in the transcription process, is called the coding strand or antisense strand. Although it is not used as a template, it has the same nucleotide sequence as the newly formed mRNA, except that thymine (T) in DNA is replaced by uracil (U) in RNA.

Steps in transcription:

1. Initiation:

The binding of DNA polymerase to DNA is a prerequisite for transcription to occur. The enzyme RNA polymerase, also known as holoenzyme, consists of the core enzyme and σ-factor

                                RNA polymerase = core enzyme + σ-factor
                                    (Holo enzyme)

Core enzyme: It consists of an assembly of peptide subunits, i.e, two α, one β, and one β’. This enzyme is responsible for the 5’-3’ RNA polymerase activity. This enzyme lacks specificity because it can’t recognise the promoter region on the DNA template.

 


Sigma factor: The sigma factor can easily be isolated from holo enzyme. It plays a significant role in promoter recognition by RNA polymerase. The γ-factor has specificity to recognize the promoter region of DNA.

Promoter region: The Promoter region is defined as the DNA sequence to which the RNA polymerase binds for the initiation of transcription. The prokaryotic promoter contains two regions;

a. Pribnow box: This is a short sequence of DNA. A stretch of six nucleotides (TATAAT). Centered approximately 10 base pairs upstream of the start of transcription.

b. 35 sequence: This is the 2nd recognition site in the promoter region, which consists of sequences TTGACA located about 35 bases left to the transcription start site.


2. Elongation:

Once the promoter region has been recognised by the holoenzyme, it begins to synthesize a transcript of DNA. Once a small portion of RNA has been formed, the σ-factor dissociates. The elongation, therefore, is carried out by the core enzyme alone. RNA polymerase doesn’t require a primer and has no known endo or exo-nuclease activity. Consequently, it has no ability to repair mistakes in the RNA as done by DNA polymerase. The unwinding activity of RNA polymerase separates the two strands over 17 base pairs. Thus, an open promoter complex is formed. The RNA polymerase (core enzyme) has 5’à3’ polymerisation activity and adds ribonucleotide triphosphate complementary to the DNA template each time during the elongation.

Chain Elongation Step:

            NTP+(NMP)n⟶(NMP)n+1+PPi

where, NTP = Ribonucleoside Triphosphate (e.g., ATP, GTP, CTP, UTP)
(NMP)n = Existing RNA chain with n nucleotides
(NMP)n+1 = Elongated RNA chain
PPi= Pyrophosphate

Pyrophosphate Hydrolysis (drives the reaction forward):

PPi⟶2Pi+Energy
where PPi = Pyrophosphate
2Pi= Inorganic phosphate 

This hydrolysis is exergonic, making the RNA synthesis reaction irreversible and energetically favorable. The resulting pyrophosphate is rapidly hydrolysed by the enzyme pyrophosphatase into two phosphate groups. The hydrolysis of pyrophosphatase releases free energy, which acts as a driving force for the polymerisation of nucleosides. As the transcription bubble moves from left to right, the DNA is unwound ahead and rewound behind as RNA is transcribed. During elongation, the growing end of the synthesized RNA strand base pairs temporarily with the DNA template to form a short hybrid RNA-DNA double helix, estimated to be 8 base pairs long. The RNA in this hybrid duplex peels up shortly after its formation, and the DNA duplex reforms.

3. Termination:

The process of elongation of the RNA chain continues until the termination signal is reached.

a) RNA polymerase can, in some instances, recognise a termination region on the DNA template (ρ-independent termination).

b) Alternatively, an additional protein, ρ factor, may be required for the release of the RNA product (ρ-dependent termination).

I. ρ dependent termination: ρ is a specific protein that binds to the growing RNA or weakly to DNA. It has ATPase activity, i.e, once attached to the transcript, it uses the energy derived from ATP hydrolysis to release the RNA from the template and from polymerase. It also dissociates RNA polymerase from DNA.

II. ρ independent termination: This is also called intrinsic termination, which consists of two sequence elements.

A short inverted repeat sequence of about 20 nucleotides, followed by a stretch of about 8 adenine-thymine (A-T) base pairs in the DNA, serves as a termination signal during transcription. These elements do not affect RNA polymerase until they are transcribed, meaning their function is carried out at the RNA level, not at the DNA level.

Once RNA polymerase transcribes the inverted repeat, the resulting RNA can fold back on itself through complementary base pairing to form a stem-loop structure, also called a hairpin. Near the base of this stem, a region rich in guanine (G) and cytosine (C) nucleotides helps stabilize the hairpin due to the strong G≡C triple hydrogen bonding.

This stable hairpin structure causes the RNA polymerase to slow down and pause. However, efficient termination only occurs when this hairpin is immediately followed by a stretch of uracil (U) residues in the RNA, which were transcribed from A residues in the DNA. A-U base pairs are the weakest among all base pairs (only two hydrogen bonds), so they are easily disrupted.

                                           

The combined effect of the stalling of RNA polymerase caused by the hairpin and the weak A-U interactions leads to destabilization of the RNA-DNA hybrid, causing the RNA transcript to dissociate from the template, thus terminating transcription.