Eukaryotic Transcription

What is Eukaryotic Transcription?

• Eukaryotic transcription is a complex process that allows eukaryotic cells to convert genetic information stored in DNA into RNA molecules, which can be transported and used for various cellular functions. This process occurs in the nucleus of eukaryotic cells, where DNA is organized into nucleosomes and higher-order chromatin structures.
• The transcription process in eukaryotes can be divided into three stages: initiation, elongation, and termination. Initiation marks the beginning of transcription, where RNA polymerase, the enzyme responsible for transcription, recognizes specific DNA sequences known as promoters and binds to them. Once bound, RNA polymerase begins to unwind the DNA double helix and initiate the synthesis of a complementary RNA strand.
• Elongation is the stage where RNA polymerase moves along the DNA template, synthesizing an RNA molecule that is complementary to the DNA sequence. This process continues until the RNA polymerase reaches a termination signal, which signals the end of transcription. Termination can occur in different ways, depending on the specific gene and regulatory factors involved.
• Eukaryotic transcription produces different types of RNA molecules with diverse functions. For instance, RNA polymerase I transcribes genes that encode structural components of the ribosome. RNA polymerase II is responsible for transcribing protein-coding genes, producing messenger RNAs (mRNAs) that carry the genetic information to the site of protein synthesis. Additionally, non-coding RNAs, which make up the majority of the transcriptional output in cells, play crucial roles in various cellular processes.
• While the basic process of transcription is similar in prokaryotes and eukaryotes, there are notable differences due to the presence of a membrane-bound nucleus and organelles in eukaryotic cells. Eukaryotes must transport their mRNA molecules from the nucleus to the cytoplasm for translation, as well as protect the mRNA from degradation. To accomplish this, eukaryotes utilize three different RNA polymerases, each responsible for transcribing a specific subset of genes.
• In summary, eukaryotic transcription is a vital process that enables cells to convert DNA information into functional RNA molecules. Through a series of stages, including initiation, elongation, and termination, RNA polymerases produce different types of RNA molecules with diverse cellular functions. Understanding the intricacies of eukaryotic transcription provides insights into gene expression regulation and the complexity of the eukaryotic genome.

Enzyme(s) Involved in Eukaryotic Transcription

Eukaryotic transcription, a crucial process for gene expression, involves the activity of multiple enzymes. These enzymes are responsible for transcribing various types of RNA molecules within the nucleus of eukaryotic cells. Unlike prokaryotes, which rely on a single RNA polymerase for RNA synthesis, eukaryotes have three distinct RNA polymerases: RNA polymerase I (RNA Pol I), RNA polymerase II (RNA Pol II), and RNA polymerase III (RNA Pol III). Let’s delve into the functions and characteristics of these enzymes involved in eukaryotic transcription.

RNA Pol I primarily operates within the nucleolus, where it transcribes specific genes responsible for producing three essential ribosomal RNA (rRNA) molecules. These rRNA molecules are the 28S, 18S, and 5.8S rRNA. Through its activity, RNA Pol I ensures the synthesis of crucial components of the ribosome, the cellular machinery responsible for protein synthesis.

Operating within the nucleoplasm, RNA Pol II takes on a central role in eukaryotic transcription. It transcribes protein-coding genes to produce precursor messenger RNA (pre-mRNA). Additionally, RNA Pol II is involved in transcribing genes that encode small nucleolar RNAs (snoRNAs) involved in rRNA processing and small nuclear RNAs (snRNAs) involved in mRNA processing, with the exception of U6 snRNA. The pre-mRNA molecules produced by RNA Pol II undergo various modifications, such as splicing and capping, before they are transported out of the nucleus for translation into proteins.

Located within the nucleoplasm, RNA Pol III transcribes genes that encode diverse RNA molecules. It synthesizes transfer RNA (tRNA), which plays a vital role in protein synthesis by bringing specific amino acids to the ribosome. Additionally, RNA Pol III transcribes the genes responsible for producing the 5S rRNA, U6 snRNA, and the 7S RNA associated with the signal recognition particle (SRP). The SRP is involved in the translocation of proteins across the endoplasmic reticulum membrane, an essential process for protein targeting.

Each of these eukaryotic RNA polymerases is composed of a large complex of 12 or more subunits. Interestingly, some of the subunits of these enzymes exhibit DNA sequence similarities to the subunits found in the core enzyme of E. coli RNA polymerase, the enzyme responsible for transcription in prokaryotes. However, a significant number of subunits in each eukaryotic RNA polymerase are unique, lacking similarities with both bacterial RNA polymerase subunits and the subunits of other eukaryotic RNA polymerases.

In conclusion, eukaryotic transcription is a complex process involving the coordinated activity of multiple enzymes. RNA polymerases I, II, and III play distinct roles in transcribing different types of RNA molecules, ranging from rRNA and pre-mRNA to tRNA and various small RNA species. Their unique subunit compositions highlight the evolutionary divergence between prokaryotic and eukaryotic transcriptional machinery. Understanding the intricacies of eukaryotic transcription and the enzymes involved is crucial for deciphering the fundamental mechanisms of gene expression in complex organisms.

Features of Eukaryotic Transcription

Eukaryotic transcription is a vital cellular process that takes place within the nucleus of eukaryotic cells. It involves the synthesis of messenger RNA (mRNA) molecules from DNA templates, which are then transported to the cytoplasm for translation into proteins. Here, we will explore the essential features of eukaryotic transcription that contribute to gene expression and protein synthesis.

1. Localization in the Nucleus: Unlike prokaryotes, eukaryotic transcription occurs exclusively within the nucleus. The DNA sequences containing the genes serve as templates for the production of RNA molecules.
2. Promoter Site Initiation: The initiation of RNA synthesis is directed by the presence of a promoter site, which is located on the 5′ side of the transcriptional start site. This region contains specific DNA sequences that provide binding sites for transcription factors and RNA polymerase.
3. Transcription by RNA Polymerase: The synthesis of RNA is catalyzed by RNA polymerase, which transcribes one strand of the DNA template known as the antisense (-) strand. This polymerase is responsible for unwinding the DNA double helix, creating a transcription bubble, and incorporating ribonucleoside triphosphates (NTPs) into the growing RNA chain.
4. Primer-Free RNA Synthesis: Unlike DNA replication, RNA synthesis does not require a primer to initiate the process. The RNA polymerase directly recognizes the promoter region and initiates transcription without the need for a separate primer molecule.
5. Directionality and Nucleophilic Attack: RNA synthesis occurs in the 5′ → 3′ direction, mirroring the direction of the DNA template strand. The RNA polymerase catalyzes a nucleophilic attack by the 3′-OH group of the growing RNA chain on the alpha-phosphorus atom of an incoming ribonucleoside 5′-triphosphate. This reaction forms a phosphodiester bond, extending the RNA chain.
6. Maturation of mRNA: In eukaryotes, the primary RNA transcript undergoes a series of modifications to produce mature mRNA. This process, known as maturation or RNA processing, involves the removal of non-coding regions called introns and the joining together of coding regions known as exons. Additionally, a modified 5′ cap and a poly(A) tail are added to the mRNA molecule, which enhance stability and facilitate translation.

In summary, eukaryotic transcription encompasses several essential features, including initiation at promoter sites, primer-free RNA synthesis, directionality, and mRNA maturation. Understanding these features provides insights into the complex process of gene expression and the regulation of protein synthesis in eukaryotic organisms.

RNA polymerase

In eukaryotes, transcription is carried out by three distinct nuclear RNA polymerases, each with specific functions and characteristics. Let’s delve into the features and roles of these RNA polymerases, shedding light on their structure and involvement in eukaryotic transcription.

1. RNA Polymerase I (Pol I): RNA Polymerase I, also known as Pol I or Pol A, is primarily located in the nucleolus. It is responsible for transcribing genes encoding larger ribosomal RNA (rRNA) molecules, specifically 28S, 18S, and 5.8S rRNAs. These rRNA genes are organized as a single transcriptional unit, resulting in a continuous precursor transcript. This precursor is later processed into the three distinct rRNA molecules. The nucleolus, a specialized structure within the nucleus, facilitates the combination of transcribed rRNAs with proteins, forming ribosomes essential for protein synthesis.
2. RNA Polymerase II (Pol II): RNA Polymerase II, referred to as Pol II or Pol B, primarily operates within the nucleus. It is responsible for transcribing various molecules, including messenger RNA (mRNA), most small nuclear RNAs (snRNAs), small interfering RNA (siRNA), and microRNA (miRNA). Precursor RNAs (pre-RNAs) generated by Pol II often exist as single-stranded transcripts, undergoing extensive processing before leaving the nucleus through nuclear pores for translation into proteins. Pre-mRNAs, for instance, undergo substantial processing steps to produce mature mRNA molecules, which are then translated in the cytoplasm.
3. RNA Polymerase III (Pol III): RNA Polymerase III, also known as Pol III or Pol C, operates within the nucleus, possibly at the nucleolus-nucleoplasm interface. Pol III transcribes small non-coding RNAs, including transfer RNA (tRNA), 5S ribosomal RNA (5S rRNA), U6 small nuclear RNA (snRNA), signal recognition particle RNA (SRP RNA), and other stable short RNAs such as ribonuclease P RNA. These small RNAs play essential roles in various cellular processes.

The three RNA polymerases in eukaryotes consist of multiple subunits. Pol I has 14 subunits, Pol II has 12 subunits, and Pol III has 17 subunits. The five core subunits of all three polymerases share homology with subunits of bacterial RNA polymerase, such as β, β’, αI, αII, and ω. Additionally, common subunits are shared among the three polymerases, while specific subunits are unique to each polymerase. Crystal structures of RNA polymerases I and II provide valuable insights into the interactions among these subunits and the intricate molecular mechanisms underlying eukaryotic transcription.

Within Pol II, the carboxyl terminal domain (CTD) of RPB1, the largest subunit, plays a crucial role in facilitating the assembly of the transcription machinery and processing of Pol II transcripts. The CTD contains repeated sequences of heptapeptides, with phosphorylation and other posttranslational modifications regulating transcription initiation, elongation, termination, and the coupling of transcription with RNA processing.

In conclusion, eukaryotic transcription relies on three distinct nuclear RNA polymerases: Pol I, responsible for rRNA synthesis; Pol II, involved in mRNA and other RNA production; and Pol III, responsible for small non-coding RNA synthesis. Each polymerase has its own subunit composition and plays a vital role in gene expression and cellular processes. Understanding the structure and functions of RNA polymerases enhances our comprehension of eukaryotic transcription at the molecular level.

Function of RNA polymerase

The genetic material in the nucleus of an Eukaryotic cell is translated through three different enzymes of RNA polymerase named RNA polymerase I II and III. What are the functions for these enzymes? Three RNA polymerases is able to transcribe distinct types of genes.

• RNA polymerase I: transcribing all the genes that make up the ribosomal RNA (rRNA) apart from the 5S rRNA.
• RNA polymerase II: It is a transcription factor that transcribes all proteins-coding genes. It is therefore responsible for the production of all mRNAs. It also transcriptionally regulates the genes that make up the majority of snRNAs, which are essential to splice RNA. It also is able to transcribe a variety of genes that make other non-coding RNAs. This includes most non-coding RNAs that are long microRNAs, snoRNAs and microRNAs.
• RNA polymerase III: It transcribed all tRNA genes, as well as five-sense rRNA genes. In a lesser degree as RNA polymerase II, it also transcribes some genes that generate other non-coding RNAs like snRNAs, microRNAs, long non-coding RNAs and snoRNAs.

Structure of an RNA Polymerase II Promoter: Key Elements and Functions

Eukaryotic promoters exhibit greater complexity and size compared to their prokaryotic counterparts, yet they share common features such as the presence of a TATA box. Let’s explore the structure of an RNA Polymerase II promoter, taking the example of the mouse thymidine kinase gene, to understand the essential elements and their functions in eukaryotic transcription.

1. TATA Box: In the mouse thymidine kinase gene promoter, the TATA box is located approximately -30 relative to the transcription initiation site (+1). The TATA box sequence in this gene is TATAAAA, read in the 5′ to 3′ direction on the nontemplate strand. Although the sequence differs from the TATA box in E. coli, it retains the A-T rich element. A-T bonds have low thermostability, facilitating local unwinding of the DNA template, preparing it for transcription.
2. CAAT Box: The mouse thymidine kinase promoter also features a conserved CAAT box (GGCCAATCT) located around -80. This sequence plays a crucial role in binding transcription factors. The CAAT box is involved in regulating gene expression by interacting with cellular factors that enhance the efficiency of transcription initiation.
3. Additional Elements: Upstream of the TATA box, eukaryotic promoters may contain other elements that contribute to gene regulation. These include GC-rich boxes (GGCG) and octamer boxes (ATTTGCAT). These elements bind specific cellular factors that promote efficient transcription initiation. They are commonly found in “active” genes that undergo continuous expression within the cell.

It’s important to note that eukaryotic promoters exhibit considerable variation in their structure and organization, depending on the specific gene and its regulatory requirements. The elements described above provide a general overview of the key components found in an RNA Polymerase II promoter.

In summary, the structure of an RNA Polymerase II promoter in eukaryotes is characterized by the presence of a TATA box, CAAT box, and other regulatory elements such as GC-rich boxes and octamer boxes. These elements play critical roles in facilitating transcription initiation, binding transcription factors, and enhancing gene expression. Understanding the structure and functions of these promoter elements contributes to our comprehension of eukaryotic transcription and gene regulation processes.

The Evolution of Promoters

While the concept of gene evolution is well-known, the evolution of promoters and other gene regulatory sequences in eukaryotes is equally significant. Mutations occurring during DNA replication can introduce changes in genes, potentially affecting the function or physical attributes of the encoded proteins. Similarly, eukaryotic promoters can undergo evolutionary modifications. Let’s delve into the evolution of promoters and how they contribute to gene expression regulation.

1. Promoter Adaptation: Consider a gene that, over numerous generations, becomes increasingly valuable to the cell. This gene might encode a structural protein crucial for a specific cellular function. In such cases, it would be advantageous for the gene’s promoter to enhance the recruitment of transcription factors and increase gene expression. Promoter adaptation plays a role in optimizing gene regulation to meet the cell’s needs.
2. Study of Promoter Sequences: Scientists studying the evolution of promoter sequences have reported varying results, partly due to the challenge of precisely defining the boundaries of eukaryotic promoters. Promoters can occur within genes, far upstream, or even downstream of the genes they regulate. However, when researchers focused on human core promoter sequences, which were experimentally identified as sequences binding the preinitiation complex, they discovered that promoters evolve at a faster pace than protein-coding genes.
3. Relationship to Organismal Evolution: The correlation between promoter evolution and the evolution of higher organisms, including humans, remains unclear. However, the evolution of promoters that modulate the expression levels of specific genes presents an intriguing alternative to the evolution of the genes themselves. Promoter adaptations can effectively increase or decrease the production of gene products, influencing cellular functions and potentially contributing to evolutionary changes.

The study of promoter evolution provides insights into the dynamic nature of gene regulation in eukaryotes. Promoters, as key regulatory elements, have the ability to evolve rapidly and adapt to changing cellular requirements. These evolutionary adaptations in promoter sequences contribute to the precise control of gene expression, ensuring the optimal synthesis of gene products necessary for cellular processes.

Transcription Factors for RNA Polymerase II

In eukaryotic transcription, the regulation of pre-mRNA synthesis goes beyond RNA polymerases and promoters. A diverse array of transcription factors, including basal transcription factors, enhancers, and silencers, play vital roles in controlling the frequency and efficiency of transcription. These factors work in conjunction to ensure the accurate synthesis of pre-mRNA from genes. Let’s explore the significance of transcription factors in RNA Polymerase II-mediated transcription.

1. Basal Transcription Factors: Basal transcription factors are essential for the assembly of a preinitiation complex on the DNA template, facilitating the recruitment of RNA Polymerase II for transcription initiation. These factors are designated with names starting with “TFII” (transcription factor for RNA Polymerase II) and are labeled from A to J. As each basal transcription factor binds to the DNA template, it further stabilizes the preinitiation complex and contributes to the efficient recruitment of RNA Polymerase II.
2. Enhancers and Silencers: Enhancers and silencers are regulatory elements that influence the efficiency of transcription but are not indispensable for the process itself. Enhancers are DNA sequences that enhance transcription when specific transcription factors bind to them. Conversely, silencers repress transcription by interacting with other regulatory proteins. These elements exert control over gene expression by modulating the rate at which transcription occurs.
3. Transcription Factor Interactions: Eukaryotic transcription involves intricate interactions among various proteins and the DNA strand. The complexity of these interactions varies for RNA Polymerases I and III, but the underlying principle remains the same. Eukaryotic transcription is tightly regulated, requiring a diverse repertoire of proteins to work together and coordinate with the DNA template. While eukaryotic transcription demands more metabolic investment compared to prokaryotes, it ensures precise transcription of pre-mRNAs necessary for protein synthesis.

The interplay between transcription factors, basal transcription factors, enhancers, and silencers is crucial for the precise control of gene expression in eukaryotic cells. These factors orchestrate the recruitment of RNA Polymerase II and the assembly of the transcription machinery, ultimately determining the transcriptional activity of specific genes.

In conclusion, transcription factors are key regulators of eukaryotic transcription, especially for RNA Polymerase II-mediated transcription. They form preinitiation complexes, enhance or repress transcription, and govern the precise synthesis of pre-mRNAs required for protein production. Understanding the roles and interactions of these transcription factors provides insights into the complex regulatory mechanisms underlying eukaryotic gene expression.

Promoter Structures for RNA Polymerases I and III

Promoters in eukaryotes display distinct characteristics depending on the RNA polymerase responsible for transcription. The promoter structures for RNA Polymerases I and III differ from each other and from those of RNA Polymerase II. Let’s explore the specific features of these promoters in eukaryotic transcription.

1. RNA Polymerase I Promoters: RNA Polymerase I is responsible for transcribing genes that encode ribosomal RNA (rRNA). These promoters contain two GC-rich sequences located in the region from -45 to +20 relative to the transcription start site. These sequences alone are sufficient for initiating transcription. However, certain genes possess additional sequences in the region from -180 to -105 upstream of the initiation site. These additional sequences further enhance the efficiency of transcription initiation by RNA Polymerase I.
2. RNA Polymerase III Promoters: RNA Polymerase III transcribes genes encoding transfer RNA (tRNA), small non-coding RNAs, and some small nuclear RNAs. Promoters for RNA Polymerase III can either be located upstream of the genes or occur within the genes themselves. These promoters are distinct from the GC-rich promoters of RNA Polymerase I. Promoters upstream of RNA Polymerase III genes often contain conserved sequence elements that are recognized by the transcription machinery specific to this polymerase.

The diversity in promoter structures for RNA Polymerases I and III reflects the variation in their transcriptional requirements and gene targets. While RNA Polymerase I promoters focus on ribosomal RNA synthesis and utilize specific GC-rich sequences, RNA Polymerase III promoters cater to the transcription of various non-coding RNAs and employ different regulatory mechanisms.

Understanding the differences in promoter structures among RNA polymerases provides insights into the specificity and regulation of eukaryotic transcription. These promoter elements guide the recruitment of the appropriate RNA polymerase to the target genes, ensuring the accurate synthesis of the required RNA molecules for cellular processes.

Components Required for Eukaryotic Transcription

RNA polymerase II

The enzyme responsible for catalyzing the linking of nucleotides in the 5′-3 direction, by using DNA for a model. The majority of eukaryotic DNA polymerase II proteins consist of 12 subunits. Two of the largest subunits have structural similarities to the b b subunits within E. coli RNA polymerase.

General transcription factors

• TFIID: TFIID is composed of TATA-binding proteins (TBP) as well as additional TBPassociated factor (TAFs). It recognizes TATA as the TATA box of promoters for eukaryotic proteins.
• TFIIB: Binds with TFIID and then allows RNA polymerase II to bind to the promoter’s core. It also promotes TFIIF binding.
• TFIIF: The TFIIF molecule binds RNA polymerase II and plays part in its capacity to attach to TFIIB and the promoter at the core. It also plays a part in the capacity for TFIIE as well as TFIIH to connect to RNA polymerase II.
• TFIIE: It plays a key role in the creation or maintaining (or either) that of the complex open. It can exert its influence through the facilitation of binding TFIIH in the presence of RNA polymerase II, and also by regulates the activities of TFIIH.
• TFIIH: Multisubunit Protein that performs multiple roles. The first is that certain subunits function as helicases, and facilitate opening of complexes. Other subunits phosphorylate carboxyl terminal domain (CTD) of RNA polymerase II and lets it interact with TFIIB which allows RNA polymerase II to move to the elongation stage.

Mediator

A multisubunit complex which mediates the effects of transcription factors that regulate on the functions of the RNA polymerase II. While mediator generally has specific subunits that are core, the composition of its subunits are different according to the type of cell and the environmental conditions. The capacity of mediator to alter the functions of RNA polymerase II is believed to happen through CTD. CTD that is a part of RNA polymerase II. Mediator could influence the capability of TFIIH to be able to phosphorylate CTD and also subunits of mediator have the capacity to make CTD. Since CTD is necessary for the release of RNA polymerase II from TFIIB Mediator plays an important role in the capacity to allow RNA polymerase II switch from the initial stage of transcription to the elongation stage in transcription.

Process of Eukaryotic Transcription

Eukaryotic transcription is a complex process that involves the synthesis of RNA from a DNA template. It occurs in the nucleus of eukaryotic cells and is divided into several stages, including initiation, elongation, and termination. Here is a step-by-step overview of the process of eukaryotic transcription:

1. Initiation:
   • Recognition and Binding: Transcription factors, proteins that regulate gene expression, recognize specific DNA sequences called promoters or enhancers in the non-coding region of the gene. These transcription factors recruit RNA polymerase II (RNAP II), the enzyme responsible for transcription.
   • Assembly of Transcription Initiation Complex: The transcription factors and RNAP II form a transcription initiation complex at the promoter region of the gene. The initiation complex includes various proteins, such as the general transcription factors, which help in stabilizing the complex and positioning RNAP II at the start site.
2. Elongation:
   • DNA Unwinding and Initiation: Once the initiation complex is formed, the DNA helix is unwound, and a small region of DNA (around 15-20 base pairs) is exposed. RNAP II synthesizes a short segment of RNA, called the abortive transcript, which is later released.
   • Promoter Clearance and Elongation: After the abortive transcript is released, RNAP II escapes the promoter region and enters the elongation phase. It starts moving along the DNA template, synthesizing an RNA molecule in the 5′ to 3′ direction. The DNA helix reforms behind the transcription bubble.
   • RNA Processing: As transcription progresses, various RNA processing events occur simultaneously. These include capping, splicing, and polyadenylation. The 5′ end of the nascent RNA molecule is modified by the addition of a 7-methylguanosine cap, which protects the RNA from degradation and helps in its export from the nucleus. Introns, non-coding regions within the gene, are removed through splicing, and the exons are joined together. Finally, a string of adenine nucleotides, called a poly(A) tail, is added to the 3′ end of the RNA.
3. Termination: Eukaryotic transcription termination is a less well-understood process compared to prokaryotes. Termination signals in eukaryotes can be polyadenylation-dependent or independent. Polyadenylation-dependent termination involves the recognition of specific sequences downstream of the gene, leading to the cleavage of the nascent RNA and the release of RNAP II. In polyadenylation-independent termination, the mechanism is less defined but involves factors that cause RNAP II to dissociate from the DNA template.
4. RNA Export: Once the RNA molecule is fully synthesized and processed, it undergoes additional modifications and is transported out of the nucleus into the cytoplasm through nuclear pore complexes. In the cytoplasm, it can undergo translation to produce proteins or perform other functions, depending on the type of RNA molecule.

It’s important to note that this is a simplified overview of eukaryotic transcription, and the process can vary depending on the specific gene and cellular context.

1. Initiation of Eukaryotic Transcription

The initiation of eukaryotic transcription marks the beginning of the synthesis of RNA from a DNA template. This process involves the recognition of specific sites on the DNA, known as promoter sites, by RNA polymerase and the assembly of various protein complexes called transcription factors. Let’s delve into the key steps and regulatory factors involved in the initiation of eukaryotic transcription.

1. Promoter Recognition and Unwinding: During initiation, RNA polymerase identifies a specific promoter site located upstream of the gene to be transcribed. The DNA in the vicinity of the promoter site is locally unwound to expose the template strand for RNA synthesis.
2. TATA Box and Initiator Element: In the case of RNA polymerase II, which transcribes protein-coding genes, most promoter sites contain a conserved sequence known as the TATA box. This sequence, typically located about 25–35 base pairs upstream of the transcription start site (+1), exhibits a consensus pattern of TATA(A/T)A(A/T). The TATA box functions similarly to the -10 sequence found in prokaryotic promoters, aiding in RNA polymerase recognition and positioning. However, certain genes lack a TATA box and instead possess an initiator element centered around the transcriptional initiation site.
3. General Transcription Factors: To initiate transcription, RNA polymerase II requires the assistance of general (or basal) transcription factors. These factors assemble into a complex on the promoter, facilitating the binding of RNA polymerase and the subsequent initiation of transcription. The general transcription factors for RNA polymerase II are collectively referred to as TFII (Transcription Factor for RNA polymerase II).
4. Formation of Transcription Initiation Complex: The first step in initiation involves the binding of the TFIID protein complex to the TATA box. TFIID contains a subunit called TBP (TATA box binding protein) that interacts with the TATA box sequence. Following TFIID binding, TFIIA stabilizes the TFIID-TATA box interaction, while TFIIB acts as a bridging protein between TFIID and RNA polymerase II. TFIIF, already complexed with RNA polymerase II, further associates with the assembly. Finally, TFIIE and TFIIH join the complex, forming the transcription initiation complex.
5. Initiation Complex Assembly: The transcription initiation complex, consisting of RNA polymerase II and the associated general transcription factors, facilitates the precise initiation of transcription. The complex includes a multitude of polypeptides and ensures the accurate positioning of RNA polymerase II on the promoter.
6. Initiator Element Binding: For genes lacking a TATA box, an initiator element substitutes the role of the TATA box in promoter recognition. Additional proteins bind to the initiator element, enabling the assembly of the transcription initiation complex in a manner similar to TATA box-containing promoters.

The initiation of eukaryotic transcription involves a complex interplay of promoter recognition, assembly of transcription factors, and the formation of the transcription initiation complex. These processes ensure the accurate initiation of RNA synthesis and the regulation of gene expression in eukaryotic cells.

2. Elongation Of Eukaryotic Transcription

Once initiation of eukaryotic transcription has taken place, the elongation phase commences, during which RNA polymerase II moves along the DNA template, synthesizing an RNA molecule in the 5′ to 3′ direction. Let’s explore the process of elongation in eukaryotic transcription and the regulatory factors involved.

1. TFIIH and Phosphorylation: TFIIH, a transcription factor complex, serves two important functions during elongation. First, it acts as a helicase, using ATP to unwind the DNA helix, facilitating the progression of transcription. Second, TFIIH phosphorylates RNA polymerase II, causing a conformational change in the enzyme and dissociation from other proteins in the initiation complex. The phosphorylation primarily occurs on the C-terminal domain (CTD) of the RNA polymerase II molecule.
2. Phosphorylation of the CTD: The CTD, a long C-terminal tail of RNA polymerase II, undergoes phosphorylation during elongation. Interestingly, RNA polymerase II with a non-phosphorylated CTD is capable of initiating transcription, but only when the CTD is phosphorylated can elongation of RNA occur. This phosphorylation event is critical for the transition from initiation to the elongation phase.
3. RNA Synthesis and Elongation: Once the RNA polymerase II has transitioned into the elongation phase, it starts moving along the DNA template, synthesizing RNA. The synthesis occurs in the 5′ to 3′ direction, with the RNA polymerase catalyzing the nucleophilic attack of the 3′-OH group of the growing RNA chain on the alpha-phosphorus atom of an incoming ribonucleoside 5′-triphosphate. This process leads to the elongation of the RNA molecule, also known as the primary transcript.
4. Modifications and Nucleosome Interaction: During elongation, additional modifications occur to the nascent RNA molecule. In eukaryotes, the 5′ end of the pre-mRNA is modified by the addition of a 7-methylguanosine (7-MG) cap. This cap structure, with an unusual 5′-5′ triphosphate linkage and methyl groups, is added when the RNA chain is approximately 30 nucleotides in length. The 7-MG cap plays a role in transcription initiation and protects the growing RNA chain from degradation by nucleases.

Elongation of transcription poses a challenge when DNA is packaged within nucleosomes, the basic units of chromatin. However, RNA polymerase II can navigate through nucleosomes with the assistance of the FACT (Facilitates Chromatin Transcription) protein complex. FACT removes histone H2A/H2B dimers from nucleosomes, leaving behind histone “hexasomes.” Once the polymerase has passed through, FACT and other proteins aid in redepositing the histone dimers, restoring the nucleosome structure.

The chromatin structure also undergoes changes depending on the activity of genes. Active genes are packaged in smaller chromatin structures and tend to have histones with more acetyl groups. In contrast, inactive genes are associated with less acetylated histones.

3. Termination of transcription in eukaryotes

The termination of eukaryotic transcription marks the final stage, resulting in the release of the complete transcript and the dissociation of RNA polymerase from the template DNA. The mechanisms of termination differ among the three types of RNA polymerases. Let’s explore the termination processes in eukaryotic transcription.

1. Factor-Dependent Termination: In the case of RNA polymerase I (Pol I), which transcribes ribosomal RNA genes, termination requires a specific transcription termination factor known as TTF-1 (Transcription Termination Factor for RNA Polymerase I). TTF-1 recognizes a specific sequence of base pairs near the end of the gene and blocks further transcription, leading to the disengagement of RNA polymerase I from the DNA template and the release of the newly synthesized RNA.

For RNA polymerase II (Pol II), responsible for transcribing protein-coding, structural RNA, and regulatory RNA genes, termination is a more complex process. As Pol II reaches the end of the gene, two protein complexes carried by the C-terminal domain (CTD) of the RNA polymerase II, CPSF (cleavage and polyadenylation specificity factor) and CSTF (cleavage stimulation factor), recognize the poly-A signal in the transcribed RNA. These complexes recruit other proteins to cleave the RNA and add a poly-A tail. The cleavage and polyadenylation process occurs at an internal site, marking the “end” of the gene, while the rest of the transcript is digested by a 5′-exonuclease. This digestion helps disengage the polymerase from the DNA template, ultimately terminating transcription.

2. Factor-Independent Termination: RNA polymerase III (Pol III) can terminate transcription efficiently without the involvement of additional factors. The termination signal for Pol III consists of a stretch of thymines located downstream from the 3′ end of mature RNAs. When Pol III encounters this poly-T termination signal, it pauses before releasing the newly synthesized RNA.

The mechanisms underlying termination are still not fully understood, and various models have been proposed. The allosteric model suggests that transcription through the termination sequence triggers conformational changes in the elongation complex, leading to termination. The torpedo model proposes that a 5′ to 3′ exonuclease degrades the second RNA strand as it emerges from the elongation complex, eventually overtaking the polymerase and causing its release.

RNA processing

RNA processing is a crucial step in the maturation of eukaryotic mRNA transcripts. These transcripts, initially referred to as heterogenous nuclear RNA (hnRNA) or pre-mRNA, undergo several processing steps to transform into mature RNA molecules that can be efficiently translated into proteins. Let’s explore the key processes involved in RNA processing.

1. Cleavage: During cleavage, larger RNA precursors are cleaved into smaller RNAs. One example is the cleavage of the primary transcript by an RNA enzyme called ribonuclease-P, which generates 5-7 tRNA precursors. This cleavage step is essential for the formation of functional tRNAs, which play a vital role in protein synthesis.
2. Capping and Tailing: Capping and tailing are modifications that occur at the ends of the primary transcript. At the 5′ end, a cap structure is added, typically consisting of 7-methyl guanosine (7 mG). This cap, derived from guanosine triphosphate (GTP), protects the mRNA from degradation and is involved in various processes, including mRNA export and translation initiation. At the 3′ end, a poly-A tail is added, which consists of a chain of adenine nucleotides. The poly-A tail contributes to mRNA stability and enhances translation efficiency.
3. Splicing: Eukaryotic primary mRNAs consist of coding exons and non-coding introns. Splicing is the process by which introns are removed, and exons are joined together to produce mature mRNA. This process is mediated by the spliceosome, a complex composed of RNA and protein molecules. The spliceosome recognizes specific sequence motifs at the boundaries between exons and introns and catalyzes the precise excision of introns. Alternative splicing allows for the generation of multiple protein isoforms from a single gene, contributing to the diversity of the proteome.
4. Nucleotide Modifications: Nucleotide modifications occur in various types of RNA molecules, such as tRNAs. These modifications include methylation (e.g., methyl cytosine, methyl guanosine), deamination (e.g., conversion of adenine to inosine), and the introduction of modified nucleotides like dihydrouracil and pseudouracil. These modifications play important roles in RNA stability, structure, and function, ultimately influencing the accuracy and efficiency of translation.

Post-transcriptional processing is essential for the conversion of primary transcripts into functional RNAs. Through cleavage, capping and tailing, splicing, and nucleotide modifications, the primary transcripts undergo a series of modifications that fine-tune their structure, stability, and functionality. These processed and matured mRNA molecules are then exported from the nucleus to the cytoplasm, where they can participate in protein synthesis through the process of translation.

Eukaryotic transcriptional control/Regulation

Eukaryotic transcriptional control, also known as regulation, plays a crucial role in gene expression in eukaryotes. It involves various levels of control that act locally to activate or suppress specific genes and globally to maintain a consistent gene expression pattern throughout the chromatin. Eukaryotic genomes are organized into compact chromatin structures, which partially conceal the DNA from the transcriptional machinery. Without regulatory proteins, many genes would be expressed at low levels or not at all. To enable transcription, the positioned nucleosomes must be displaced, allowing the transcriptional machinery access to the DNA.

Transcription initiation is the primary level at which gene expression is regulated. It involves cis-acting elements (enhancers, silencers, isolators) within the regulatory regions of the DNA, as well as sequence-specific trans-acting factors acting as activators or repressors. Additionally, transcription can be regulated post-initiation by controlling the movement of the elongating polymerase.

• Global control and epigenetic regulation: The global control of gene expression in eukaryotes involves the regulation of chromatin structure. The chromatin can be either “open” and transcriptionally permissive (euchromatin) or “condensed” and transcriptionally inactive (heterochromatin). Transcription can be silenced through histone modification, RNA interference, and DNA methylation. These modifications affect the accessibility of DNA and play a role in the inheritance of gene expression patterns through cell division, a process known as epigenetic regulation.
• Gene-specific activation: Gene-specific activation is achieved through diverse mechanisms that override inhibitory signals at the gene promoter. Eukaryotic genes contain extensive regulatory sequences with multiple regulator-binding sites spread throughout kilobases from the promoter. Enhancers, clusters of regulator binding sites, facilitate cooperative action among transcription factors. Activators recruit the transcriptional machinery directly or other factors required for transcription initiation. They can also recruit nucleosome modifiers to alter chromatin structure and facilitate transcription.
• Gene-specific repression: Gene-specific repression involves inhibiting the function of transcriptional activators. Repressors can overlap with activator binding sites, preventing activator binding, or inhibit activators through various mechanisms such as masking activating domains or promoting degradation. Repressors can directly interact with the transcriptional machinery or recruit histone modifiers and nucleosome remodeling enzymes to repress transcription. These mechanisms can lead to transcriptional silencing that spreads along the chromatin, affecting multiple genes.
• Elongation and termination control: Elongation and termination control are also important aspects of transcriptional regulation. During elongation, RNA polymerase moves along the DNA strand until it reaches a termination sequence. Elongation can be regulated by factors such as the transcriptional activator Tat, which affects elongation rather than initiation. Pausing of polymerase during elongation can influence chromatin structure and lead to rapid transcriptional responses. Transcription termination is coupled with efficient polymerase recycling and can influence gene looping and re-initiation efficiency.

In summary, eukaryotic transcriptional control is a complex process involving multiple levels of regulation. It includes the regulation of chromatin structure, gene-specific activation and repression, and control of elongation and termination. These mechanisms ensure precise gene expression patterns and play a crucial role in shaping cell identity and responding to cellular needs.

What Is RNA Editing?

In accordance with the fundamental dogma of molecular biology, information about genetics is transferred from DNA to proteins during the process of the expression of genes. The genetic information does not change within the mRNA intermediary. However, the development of RNA editing has revealed that it is possible to have exceptions. Editing RNA processes alter the gene’s information content transcripts by two methods: 

1. by changing the structures of individual bases and 
2. by inserting or deleting uridine monophosphate residues.

RNA Editing by changing the structures of individual bases

The first kind of RNA editing that results in the substitution of one base with another, is a rare phenomenon. This kind of editing was found in research on the apolipoprotein B (apo-B) gene and the mRNAs found in rabbits as well as humans. Apolipoproteins are blood protein which transport specific types of fat molecules within circulation. Within the liver, apoB gene encodes a huge protein that is 4563 amino acids long. In the intestines, the apo-B is mRNA that controls the synthesis of a protein that is only 2153 amino acid long. In this case, an C residue from the pre-mRNA gets converted into an U which creates an internal UAA translation-termination codon that results in the apolipoprotein being shortened.

UAA is one codon which end polypeptide chains that are undergoing translation. If there is a UAA codon is created within the coding area of an mRNA it will prematurely end the polypeptide’s translation process which results in an unfinished gene product. The C U conversion process is catalyzed by a sequence-specific RNA binding protein, which eliminates amino groups from the cytosine sequences. Similar instances in RNA editing is reported for an mRNA identifying the protein (the glutamate receptor) that is found in rat brain cells. A more extensive editing of mRNAs of the C type and the U kind occurs inside the plants’ mitochondria where the majority transcripts of genes are altered to a certain degree. Mitochondria possess unique DNA genomes, as well as the machinery for protein synthesizing. There are a few transcripts that can be found in plant mitochondria, the majority of the C’s get converted into U residues.

RNA Editing by inserting or deleting uridine monophosphate residues

Another, more complicated form of RNA editing can be found within the mitochondria in Trypanosomes (a group of flagellated protozoa which cause sleepiness in humans). In this instance the uridine monophosphate molecule is added to (occasionally removed from) genes, causing significant changes in the polypeptides identified by mRNA molecules. The editing of RNA is controlled through guide RNAs that have been transcribed from different mitochondrial genes. Guide RNAs have sequences that are partly identical to the pre-mRNAs which can edit. The pairing between guide RNAs as well as the pre-mRNAs causes gaps that have non-paired A residues in guides RNAs. Guide RNAs are used as templates to edit, since U’s are placed into the gaps in premRNA molecules that are opposite to those in guides RNAs.

Why do these RNA editing processes occur?

What are the nucleotide sequences for these mRNAs not outlined in the mitochondrial genes like they are found in many nuclear genes? So far, the solutions to these intriguing questions are only speculations. Trypanosomes are eukaryotes that were single-celled that separated from other eukaryotes earlier in their evolution. Many evolutionary scientists have suggested the possibility that editing RNA was prevalent in the early cells, and several reactions are believed to be catalyzed through proteins, not RNA molecules. Another theory suggests the idea that RNA editing is an ancient method for changing the patterns that regulate gene expression. No matter the reason it is believed that RNA editing is a key factor for the expression of the genes within mitochondria in trypanosomes as well as plants.

Eukaryotic Genes Have a Core Promoter and Regulatory Elements

To ensure that transcription occurs at a suitable rate the eukaryotic genes possess two essential elements: a primary promoter as well as regulatory elements. The image illustrates a common pattern of sequences that are found in proteins-encoding genes. The promoter’s primary function is to be a DNA sequence that is relatively short that is essential to allow transcription to occur. It is typically comprised of the TATAAA sequence, which is known as the TATA box, and the transcriptional start point, which is where transcription starts.

The TATA box that is typically 25 bp further downstream than the transcriptional start point and is essential in determining the precise start location for transcription. If it’s missing from the promoter that is the core the location of the transcription start is unclear and transcription can begin in a variety of places. The core promoter by itself is responsible for a low degree of transcription. This is referred to as basal transcription.

The regulatory elements are short DNA sequences that impact the capacity that RNA polymerase can recognize a core promoter, and then begin with transcription. They are identified by transcription factors, proteins that affect the speed of transcription.

There are two types in the regulatory element category. Activating sequences, also known as enhancers are essential to trigger transcription. Without enhancer sequences most genes in the eukaryotic family have low levels of transcription. In certain circumstances it is possible to block transcription of a specific gene.

Silencers are DNA sequences which are identified by transcription factors that hinder transcription. The most frequent location of regulatory factors is in the -50 to -100 area. However, the location of these elements differ across different eukaryotic gene types. They can be located far away from the promoter’s core but they can significantly influence the capacity of RNA polymerase to start transcription.

DNA sequences, such as those in the TATA box and enhancers and silencers work only on a specific gene. They are referred to as cis-acting components. The word “cis” is derived from the chemistry nomenclature which means “next to.” Cis-acting elements, although they could be away from the promoter’s core but are always located on the same chromosome as genes they control. Contrary to this the transcription factors that regulate that bind to these elements are known as trans-acting factor (the term trans translates to “across from”).

Transcriptional factors which regulate the expression of genes are encoded by genes. Regulatory genes which encode transcription factors can be different from the genes they regulate or even on an entirely different chromosome. If a gene that encodes an trans-acting protein is expressed, the transcription protein may be released into the cell nucleus and attach to its appropriate cis-acting component. Now let’s turn our attention to the role of these proteins.

Significance of Transcription

Transcription is a process that involves the conversion of genetic information stored in DNA into an RNA molecule. It plays a crucial role in gene expression and is a fundamental process in biology with significant implications. Here are some key points highlighting the significance of transcription:

1. Gene Expression: Transcription is the first step in the process of gene expression, where the genetic information encoded in the DNA is transcribed into RNA. This RNA molecule, known as messenger RNA (mRNA), serves as a template for protein synthesis during translation. Thus, transcription allows the transfer of genetic information from DNA to RNA, enabling the production of proteins that are essential for cell structure, function, and regulation.
2. Regulation of Gene Expression: Transcription serves as a crucial regulatory step in controlling gene expression. It allows cells to selectively transcribe specific genes in response to various signals, developmental cues, and environmental factors. Transcription factors and other regulatory proteins bind to specific DNA sequences near the gene, either enhancing or inhibiting the transcription process. By modulating transcription, cells can control the production of different proteins and thereby regulate their functions.
3. Cell Differentiation and Development: Transcription plays a vital role in cellular differentiation and development. During embryonic development, different cell types arise from a single fertilized egg. This process is regulated by the selective transcription of specific genes, leading to the expression of distinct proteins in different cell types. Transcription factors and other regulatory molecules orchestrate these processes, allowing cells to adopt specific fates and acquire specialized functions.
4. Disease and Pathogenesis: Transcriptional dysregulation can lead to the development of various diseases. Mutations or alterations in the transcriptional machinery, regulatory elements, or transcription factor binding sites can disrupt normal gene expression patterns. Such abnormalities can contribute to the development of genetic disorders, cancer, metabolic diseases, and other pathological conditions. Understanding transcriptional processes and their regulation is essential for deciphering the molecular mechanisms underlying diseases and developing targeted therapies.
5. Research and Biotechnology: Transcription is a crucial tool in research and biotechnology. Techniques such as reverse transcription polymerase chain reaction (RT-PCR) allow the amplification and detection of specific RNA molecules, enabling gene expression profiling, diagnosis of diseases, and monitoring treatment responses. Transcriptional profiling using microarrays or next-generation sequencing technologies provides insights into global gene expression patterns and helps identify differentially expressed genes in various conditions. Moreover, the development of gene editing technologies like CRISPR-Cas9 relies on understanding the transcriptional processes involved in DNA repair mechanisms.

Difference between prokaryotic and eukaryotic transcription – Transcription in prokaryotes vs eukaryotes

Aspect	Prokaryotic Transcription	Eukaryotic Transcription
Location	Cytoplasm	Nucleus
RNA Release and Proceeding	Cytoplasm	Nucleus
RNA Polymerase Polypeptides	Five	Ten to Fifteen
Simultaneous Transcription and Translation	Yes	No
Complexity	Less complex	More complex
RNA Polymerase Types	One	Three
Promoter Variation	Less	More
Post-Transcriptional Modifications	Absent	Present
mRNA Sequence	Polycistronic	Monocistronic

FAQ

References

• https://openoregon.pressbooks.pub/mhccmajorsbio/chapter/eukaryotic-transcription/
• https://www.tutorialspoint.com/difference-between-prokaryotic-and-eukaryotic-transcription
• https://courses.lumenlearning.com/suny-biology1/chapter/eukaryotic-transcription/
• https://www.cell.com/current-biology/pdf/S0960-9822(08)01569-8.pdf
• https://www.coursehero.com/study-guides/boundless-biology/eukaryotic-transcription/
• https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/eukaryotic-genes/eukaryotic-transcriptional-control