Transcription 

Genetic information expression comprises transcription and translation. In transcription, a DNA gene strand serves as a template to synthesise a complementary RNA strand, forming the gene transcript. For instance, the DNA sequence AAA generates the complementary RNA sequence UUU. Translation, on the other hand, converts the RNA transcript's nucleotide sequence into the amino acid sequence of the polypeptide gene product. Transcription plays a pivotal role in transcribing genetic information from DNA to RNA, serving as a crucial step in gene expression.

1.0Transcription Definition

The genetic information in the nucleotide sequence of DNA is passed on for protein synthesis through an intermediate messenger RNA (mRNA). A mRNA is a complementary copy of one of the two DNA strands that make up a gene. The formation of an RNA copy from a DNA template is called transcription. 

2.0What is Transcription? 

Transcription is the vital process through which genetic instructions encoded within DNA are transcribed into RNA molecules. Only one of the two DNA strands, known as the antisense or template strand, serves as the template for transcription. RNA polymerase reads this strand from the 3' to 5' direction, synthesising a complementary RNA strand in the 5' to 3' direction, except with uracil replacing thymine. This directionality arises because RNA polymerase can only add nucleotides to the 3' end of the growing mRNA chain. 

The complementary RNA, also called the transcript, mirrors the sequence of the sense or non-template (coding) strand of DNA. The RNA polymerase enzyme initiates transcription by binding to the DNA template, unwinding the DNA strands, and catalysing the synthesis of the RNA strand using RNA nucleotides. The resulting RNA transcript plays essential roles within the cell, such as serving as templates for protein synthesis or functioning as regulatory non-coding RNAs. Transcription is a fundamental process in gene expression, crucial for cellular function and regulation.

3.0RNA Polymerase

RNA polymerase is an enzyme responsible for catalysing the synthesis of RNA from a DNA template during the process of transcription. It plays a central role in gene expression by transcribing the genetic information encoded in DNA into RNA molecules. RNA polymerase binds to specific regions of DNA called promoters, unwinds the DNA strands, and synthesises an RNA molecule complementary to one of the DNA strands. 

RNA polymerase is a holoenzyme that is represented by (𝛂𝛂𝛃𝛃'𝛚)σ. While the core enzyme (𝛂𝛂𝛃𝛃'𝛚) can transcribe DNA into RNA, transcription can begin nonspecifically at any base on the DNA without the aid of additional factors.

• Sigma Factor (σ): This subunit imparts specificity to the transcription process. It binds to the promoter region of DNA, thereby initiating transcription.
• Core Complex: This component is responsible for the continuation of transcription once it has been initiated.
• Rho Factor (ρ): This factor is essential for the termination of transcription, ensuring that the process concludes at the appropriate point.

4.0Transcription Unit

A transcription unit in DNA is defined primarily by the three regions in the DNA: 

(i) A Promoter 

(ii) The Structural gene 

(iii) A Terminator

There is a convention in defining the two strands of the DNA in the structural gene of a transcription unit. Since the two strands have opposite polarity and the DNA-dependent RNA polymerase also catalyse the polymerisation in only one direction, that is, 5'→3', the strand that has the polarity 3'→5' acts as a template, and is also referred to as template strand. The other strand which has the polarity (5'→3') and the sequence same as RNA (except thymine at the place of uracil), is displaced during transcription. Strangely, this strand (which does not code for anything) is referred to as coding strand. All the reference point while defining a transcription unit is made with coding strand.

The promoter and terminator flank the structural gene in a transcription unit. The promoter is said to be located towards 5' -end (upstream) of the structural gene (the reference is made with respect to the polarity of coding strand). It is a DNA sequence that provides binding site for RNA polymerase, and it is the presence of a promoter in a transcription unit that also defines the template and coding strands. By switching its position with terminator, the definition of coding and template strands could be reversed. 

The terminator is located towards 3' -end (downstream) of the coding strand and it usually defines the end of the process of transcription (Figure 5.9). There are additional regulatory sequences that may be present further upstream or downstream to the promoter. Some of the properties of these sequences shall be discussed while dealing with regulation of gene expression.

5.0Transcription Unit and the Gene

• A gene is defined as the functional unit of inheritance. Though there is no ambiguity that the genes are located on the DNA, it is difficult to literally define a gene in terms of DNA sequence. The DNA sequence coding for tRNA or rRNA molecule also define a gene. However by defining a cistron as a segment of DNA coding for a polypeptide, the structural gene in a transcription unit could be said as monocistronic (mostly in eukaryotes) or polycistronic (mostly in bacteria or prokaryotes). 
• In eukaryotes, the monocistronic structural genes have interrupted coding sequences – the genes in eukaryotes are split. The coding sequences or expressed sequences are defined as exons. Exons are said to be those sequence that appear in mature or processed RNA. The exons are interrupted by introns. Introns or intervening sequences do not appear in mature or processed RNA. 
• The split-gene arrangement further complicates the definition of a gene in terms of a DNA segment. Inheritance of a character is also affected by promoter and regulatory sequences of a structural gene. Hence, sometime the regulatory sequences are loosely defined as regulatory genes, even though these sequences do not code for any RNA or protein. 

6.0Stages of Transcription

The process of transcription can be divided into three stages: 

(A) Initiation 

(B) Elongation 

(C) Termination

Initiation

• Promoter Recognition: Transcription begins when RNA polymerase, the enzyme responsible for synthesising RNA from the DNA template, binds to a specific sequence of DNA known as the promoter. This region is located at the beginning of a gene.
• Formation of the Transcription Bubble: The binding of RNA polymerase to the promoter causes the DNA to unwind, forming a transcription bubble. This unwinding exposes the template strand of DNA that will be transcribed into RNA.
• Start Site Selection: Within the promoter, a specific start site is recognized where transcription of the gene into RNA will actually begin.
• Sigma Factor and Core Enzyme: The sigma factor (σ) is a component of the holoenzyme that specifically recognizes the promoter sequences. After the initial RNA synthesis, the sigma factor dissociates from the polymerase, leaving the core enzyme to continue RNA synthesis.

Elongation

• RNA Chain Elongation: RNA polymerase moves along the DNA template strand, adding ribonucleotides (RNA building blocks) in the 5' to 3' direction (that is, it reads the template strand in the 3' to 5' direction). The ribonucleotides are added according to the rules of complementary base pairing (A pairs with U, and C pairs with G in RNA).
• Transcription Bubble Movement: The transcription bubble moves with the RNA polymerase, unwinding the DNA ahead of the enzyme and rewinding it behind. This process ensures that only the part of the DNA being actively transcribed is unwound at any given time.

Termination

• Signal Recognition: Termination of transcription occurs when RNA polymerase encounters a termination signal in the DNA sequence. This signal varies among organisms and can involve different mechanisms.
• RNA Polymerase Release: In prokaryotes, the termination signal often forms a hairpin structure in the RNA that causes RNA polymerase to detach from the DNA, releasing the newly synthesised RNA.

• In E. coli, transcription termination occurs via rho-dependent and rho-independent mechanisms.
• Rho-independent termination involves a GC-rich region with inverted repeats on the DNA, leading to the formation of a hairpin structure in the RNA, which causes RNA polymerase to pause and the nascent RNA molecule to detach due to weak AU base pairs.
• Rho-dependent termination also relies on a hairpin structure to pause RNA polymerase but requires the additional action of the rho protein. The rho protein binds to an RNA sequence called the rut site, follows RNA polymerase, and uses its helicase activity to separate the RNA transcript from the DNA template upon encountering the paused polymerase at the hairpin.

7.0Transcription in Eukaryotes and RNA splicing

The primary transcript is converted into functional mRNA through post-transcriptional processing, which involves three key steps:

A. Modification of the 5' End by Capping: The capping of the 5' end occurs rapidly after the start of transcription. A methylated guanosine (with a methyl group at the 7th position) is added at the 5' end with the help of the enzyme guanyl transferase. This cap is crucial for the formation of the mRNA-ribosome complex. Translation is not possible if the cap is missing because the cap is recognized by the 18S rRNA of the ribosomal unit.

B. Polyadenylation at the 3' End (Tailing): Polyadenylation involves the addition of a poly (A) tail to the 3' end of the newly formed hnRNA, facilitated by the enzyme Poly (A) polymerase. This process adds about 200-300 adenine residues.

C. Splicing of hnRNA (Tailoring): In eukaryotes, the coding sequences of RNA (exons) are interrupted by non-coding sequences (introns). A small nuclear RNA (snRNA) and protein complex called small nuclear ribonucleoproteins (snRNPs, or "snurps") play a crucial role in this process. During splicing, introns are removed, and exons are joined together, producing mature mRNA.

Typically, mRNA in eukaryotes carries the codons for a single complete protein molecule (monocistronic mRNA). In contrast, in prokaryotes, mRNA carries codons from several adjacent DNA cistrons, resulting in a much longer molecule (polycistronic mRNA).