DNA transcription is the biological process by which an RNA molecule is synthesized using a DNA template under the direction of DNA and the genetic information is transferred to the RNA.
Place of DNA Transcription
Prokaryotes lack a distinct nucleus so the DNA transcription process takes place in the cytoplasm. The entire process occurs in the vicinity of the DNA molecule, with the RNA synthesis initiated directly on the DNA template.
In eukaryotes, DNA transcription occurs in the nucleus. The DNA is first translocated from the nucleus to the cytoplasm as precursor mRNA (pre-mRNA).
A segment of DNA that is transcribed into a single RNA molecule is referred to as a transcriptional unit.
In prokaryotes, a transcriptional unit typically corresponds to a single gene. The RNA polymerase binds to the promoter region and transcribes the entire gene sequence into a single mRNA molecule.
In eukaryotes, transcriptional units can be more complex due to the presence of introns and alternative splicing. Multiple genes can be organized within a single transcriptional unit, and different alternative splicing patterns can generate multiple mRNA isoforms from a single gene.
The transcriptional unit mainly consists of three regions-
- Structural gene
A promoter is a region of DNA located upstream of a gene that plays a crucial role in initiating the process of DNA transcription. It is responsible for regulating the transcription process.
Transcription factors, which are proteins that attach to the promoter and start the DNA transcription process, can recognize certain DNA sequences in promoters.
Depending on the specific regulatory elements present in the promoter region, these transcription factors can either stimulate or inhibit gene expression.
The promoter region typically includes several components, such as the core promoter, proximal promoter, and distal enhancers.
(a) Core Promoter
The core promoter contains the transcription start site (TSS) where RNA polymerase binds and begins the transcription process.
(b) Proximal Promoter
The proximal promoter region is located immediately upstream of the core promoter and contains binding sites for various transcription factors.
(c) Distal Enhancers
The distal enhancers are additional regulatory elements located farther away from the gene and can influence gene expression by interacting with the promoter region.
The sequence and structure of the promoter region determine the strength and specificity of gene expression. Different genes have distinct promoter regions, allowing for the precise control of gene expression in response to various signals, developmental stages, and environmental conditions.
2. Structural Gene
A structural gene is a specific type of gene that contains the instructions for synthesizing a functional protein or RNA molecule. These genes encode the primary structure of a protein, which is essential for carrying out various cellular functions.
It’s important to note that not all genes are structural genes. There are other types of genes, such as regulatory genes, that control the expression of structural genes. Regulatory genes encode proteins or RNA molecules that interact with the promoter regions or other regulatory elements to modulate the transcription of structural genes. Together, structural and regulatory genes play crucial roles in the functioning and development of living organisms.
the structural gene consists of coding and template strands. Let’s explore these concepts:
(a) Coding Strand:
The coding strand, also known as the sense strand or non-template strand, is the DNA strand that has the same sequence as the RNA transcript (with T’s replaced by U’s in RNA).
(b) Template Strand
The template strand, also known as the antisense strand or non-coding strand, is the DNA strand used as a template during the synthesis of RNA. The RNA molecule is synthesized complementary to the template strand, so its sequence is the reverse complement of the template strand.
A terminator refers to a specific DNA sequence that marks the end of a gene or a transcription unit. It is also known as a transcription terminator or transcription termination site.
During DNA transcription, RNA polymerase moves along the DNA template strand, synthesizing an RNA molecule in the 5′ to 3′ direction. At a termination site, the terminator sequence signals the RNA polymerase to stop transcription and release the newly synthesized RNA molecule.
Terminator sequences typically consist of specific DNA sequences that form secondary structures, such as hairpin loops or stem-loop structures, in the RNA molecule. These structures cause the RNA polymerase to pause and destabilize the transcription complex, leading to its dissociation from the DNA template.
There are two main types of transcription terminators:
- Rho-independent terminators: These terminators rely on the specific DNA sequence and the formation of a stem-loop structure in the RNA transcript. The stem-loop structure followed by a stretch of uracil residues (in the DNA template strand) causes the RNA polymerase to stall and eventually dissociate from the DNA, terminating transcription.
- Rho-dependent terminators: These terminators require the action of a protein called the Rho factor. Rho factor recognizes specific sequences in the RNA transcript and interacts with the RNA polymerase, leading to its dissociation and termination of transcription.
Steps of DNA Transcription
DNA transcription involves three steps that can be summarized as follows:
The first step is initiation, where the enzyme RNA polymerase, binds to the DNA template strand at the promoter. The promoter contains specific DNA sequences recognized by the RNA polymerase and other regulatory proteins.
Once the RNA polymerase is bound to the promoter, it unwinds a small section of the DNA helix to expose the template strand.
Initiation factors are proteins that play a crucial role in the initiation of DNA transcription.
These factors are involved in the assembly and activation of the transcription initiation complex, which includes RNA polymerase and other regulatory proteins.
They help ensure that transcription starts at the correct location and under appropriate conditions.
Here are some important initiation factors involved in transcription in eukaryotes:
A. Transcription factor IID (TFIID): TFIID is one of the key initiation factors in eukaryotic DNA transcription. It is composed of several subunits, including TATA-binding protein (TBP), which recognizes and binds to the TATA box sequence in the promoter region of genes.
TFIID serves as a scaffold for the assembly of other transcription factors and RNA polymerase at the promoter, initiating the formation of the preinitiation complex.
B. Transcription factor IIA (TFIIA): TFIIA is another initiation factor that interacts with TFIID and stabilizes its binding to the promoter. It helps in positioning and stabilizing RNA polymerase at the transcription start site.
C. Transcription factor IIB (TFIIB): TFIIB plays a crucial role in the recruitment of RNA polymerase to the promoter and the positioning of the transcription start site. It interacts with both TFIID and RNA polymerase, facilitating the assembly of the preinitiation complex.
D. Transcription factor IIH (TFIIH): TFIIH is a multi-subunit complex involved in multiple aspects of transcription. It has helicase activity, which unwinds the DNA double helix near the transcription start site, creating a transcription bubble.
TFIIH also possesses kinase activity, phosphorylating the C-terminal domain (CTD) of RNA polymerase II, leading to the transition from initiation to the elongation phase of DNA transcription.
E. Mediator complex: The Mediator complex is a large protein complex that acts as a bridge between transcription factors, RNA polymerase, and the promoter region. It facilitates communication and coordination between these components to regulate transcriptional initiation and response to various signals.
DNA Transcription: Steps, Places & Transcriptional Unit
After initiation, RNA polymerase moves along the DNA template strand in the 3′ to 5′ direction, synthesizing an RNA molecule in the 5′ to 3′ direction. The enzyme catalyzes the addition of complementary RNA nucleotides to the growing RNA chain, using the DNA template strand as a guide. The DNA helix re-forms after the RNA polymerase passes, and the newly synthesized RNA molecule is elongated.
The final step is termination, where RNA polymerase reaches a termination site on the DNA template strand. In bacteria, termination can occur through two mechanisms: Rho-dependent termination and Rho-independent termination.
In Rho-dependent termination, a protein called Rho factor interacts with the RNA transcript and the RNA polymerase, causing the enzyme to dissociate from the DNA template.
In Rho-independent termination, specific sequences in the RNA transcript form a stem-loop structure followed by a stretch of uracil residues, leading to the release of the RNA transcript and the dissociation of the RNA polymerase.
Termination factors are proteins involved in the termination of DNA transcription, the process by which RNA synthesis is completed and the RNA molecule is released from the DNA template. These factors play a crucial role in recognizing termination signals and facilitating the dissociation of RNA polymerase from the DNA template. Here are some important termination factors:
1. Rho Factor: Rho factor is a termination factor found in bacteria. It is an ATP-dependent helicase protein that binds to specific termination sequences in the RNA transcript.
Rho factor interacts with the RNA polymerase complex and uses its helicase activity to unwind the RNA-DNA hybrid, leading to the release of the RNA molecule and termination of transcription.
2. Rho-independent Termination Factors: In bacteria, certain termination signals can lead to transcription termination without the involvement of the Rho factor. These sequences are commonly referred to as Rho-independent or intrinsic terminators.
They consist of two key elements: a GC-rich region followed by a stretch of adenine residues in the DNA template strand. During transcription, the GC-rich region in the RNA transcript forms a stable stem-loop structure, followed by a series of uracil residues. This structure causes RNA polymerase to pause and destabilizes the transcription complex, leading to its dissociation and termination of DNA transcription.
3. Termination-associated Proteins:
In eukaryotes, the termination of DNA transcription involves the interplay of multiple factors. One such factor is the cleavage and polyadenylation specificity factor (CPSF), which recognizes and binds to the polyadenylation signal in the pre-mRNA molecule.
CPSF promotes the cleavage of the pre-mRNA at a specific site and subsequently facilitates the addition of a poly(A) tail to the mRNA. Other termination-associated proteins, such as cleavage stimulation factor (CstF) and poly(A) polymerase, are also involved in the process of transcription termination and mRNA processing.
It’s important to note that termination mechanisms can vary between bacteria and eukaryotes, and different factors may be involved in each system.
Tata Box and GC Box
The Tata box and GC box are both regulatory elements involved in the initiation of DNA transcription.
1. Tata Box
The Tata box, also known as the TATA box or Goldberg-Hogness box, is a DNA sequence found in the promoter region of many eukaryotic genes. It is named after its highly conserved sequence “TATAAA” or a slight variation thereof (e.g., TATAAT). The Tata box is typically located 25–35 base pairs upstream of the transcription start site.
In prokaryotes, 10 bp upstream from, the start point lies a conserved sequence described as –10 sequence TATAAT or “Pribnow box” and –35 sequence TTGACA as “Recognition sequence”.
upstream (opposite to the direction of transcription) or downstream (in the same direction as transcription)
The Tata box serves as a binding site for the transcription factor known as TATA-binding protein (TBP). TBP is a subunit of the TFIID transcription factor complex, which is responsible for recognizing and binding to the Tata box. Once TBP binds to the Tata box, it recruits other transcription factors and RNA polymerase to the promoter region, facilitating the assembly of the pre-initiation complex and the initiation of transcription.
2. GC Box
The GC box is also known as the GC-rich box or Sp1 binding site. It is another regulatory element found in the promoter region of genes.
It is characterized by a high density of guanine (G) and cytosine (C) nucleotides and has the consensus sequence 5′-GGGCGG-3′ or 5′-GGGCGGG-3′. The GC box is often located proximal to the Tata box or near other regulatory elements.
CG-rich stretch of 20–50 nucleotides present on DNA within ≈100 base pairs upstream of the start-site region known as GC box.
The function of the GC box is that it is recognized and bound by a transcription factor called specificity protein 1 (Sp1).
Sp1 is a zinc finger protein that binds to the GC-rich sequences in DNA. Sp1 binding to the GC box can enhance gene transcription by promoting the assembly of the transcription initiation complex and recruiting additional transcriptional co-activators.
The Tata box and GC box both are examples of cis-regulatory elements that influence gene expression.
RNA Polymerase Enzyme
RNA polymerases are multisubunit complexes composed of several subunits that work together to carry out DNA transcription. RNA polymerase is a holoenzyme that is represented as (a2bb’w)s and the enzyme without s subunit is referred to as the core enzyme.
Bacterial RNA Polymerase (RNAP)
1. Core Enzyme:
– α subunits (2-3 copies): Involved in assembly, stability, and interaction with regulatory factors.
– β subunit: Catalytic subunit responsible for polymerization.
– β’ subunit: Involved in DNA binding and structural integrity.
– ω subunit: Essential for assembly and stability of the core enzyme.
2. Sigma Factor:
– σ subunit: Recognizes and binds to specific DNA sequences (promoters) to initiate transcription.
Eukaryotic RNA Polymerase II (RNAPII):
1. Core Enzyme:
– Rpb1 subunit: Largest subunit with the catalytic activity responsible for polymerization.
– Rpb2 subunit: Involved in binding the DNA template and RNA product.
– Rpb3, Rpb4, Rpb5 subunits: Stabilize the core enzyme and assist in binding factors.
– Rpb6 subunit: Essential for enzyme assembly and stability.
2. General Transcription Factors:
– TFIIB, TFIID, TFIIE, TFIIF, TFIIH: Assist in the initiation, elongation, and termination of DNA transcription by interacting with the core enzyme and DNA.
In eukaryotes, the RNA polymerases are of three types–
- RNA polymerase I
- RNA polymerase II
- RNA polymerase III.
Functions of different RNA polymerases in eukaryotes-
(i) RNA polymerase-I → 5.8 S, 18 S, 28 S rRNA synthesis
(ii) RNA polymerase-II → Hn RNA (heterogeneous nuclear RNA), mRNA synthesis
(iii) RNA polymerase-III → tRNA, Sc RNA (small cytoplasmic RNA), 5S rRNA and Sn RNA (small nuclear RNA) synthesis.
DNA Transcription: Steps, Places & Transcriptional Unit