Eukaryotic Transcription

Transcription is the process of copying genetic information from DNA into RNA, especially mRNA, by the enzyme RNA polymerase. It is the first step of gene expression, in which a particular segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA polymerase. It results in a complementary, antiparallel RNA strand called a primary transcript.

Transcription in eukaryotes is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Eukaryotic transcription is carried out in the nucleus of the cell by one of three RNA polymerases, depending on the RNA being transcribed, and proceeds in three sequential stages: initiation, elongation, and termination .

Initiation requires the binding of transcription factors to promoter sequences upstream of the gene . The most-extensively studied core promoter element in eukaryotes is a short DNA sequence known as a TATA box, found 25-30 base pairs upstream from the start site of transcription. The TATA box is recognized by a transcription factor called TATA-binding protein (TBP), which is part of a larger complex called TFIID. Other transcription factors and RNA polymerase then assemble on the promoter to form a pre-initiation complex (PIC). In addition, transcription is also regulated by upstream control elements that lie 5′ to the TATA box.

Elongation involves the synthesis of RNA by RNA polymerase along the DNA template. The RNA polymerase transcribes one strand, the antisense (-) strand, of the DNA template. RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on an incoming ribonucleoside 5-triphosphate.

Termination occurs when RNA polymerase reaches a termination signal on the DNA template and releases the RNA transcript. Unlike prokaryotes, eukaryotes do not have specific termination sequences on their genes. Instead, termination is coupled with polyadenylation, a process that adds a poly-A tail to the 3` end of the mRNA transcript. A cleavage and polyadenylation specificity factor (CPSF) recognizes a polyadenylation signal (AAUAAA) on the pre-mRNA and cleaves it downstream of this signal. A poly-A polymerase then adds about 200 adenine nucleotides to form the poly-A tail.

The primary transcript undergoes various processing steps before becoming a mature mRNA that can be exported to the cytoplasm for translation. These steps include capping, splicing, and editing. Capping involves adding a modified guanine nucleotide (7-methylguanosine) to the 5` end of the pre-mRNA. This cap protects the mRNA from degradation and facilitates its transport and recognition by the ribosome. Splicing involves removing non-coding introns and joining coding exons to produce a continuous coding sequence. This is done by a complex of proteins and RNAs called spliceosome. Editing involves changing some nucleotides in the pre-mRNA to alter its sequence and function. This can be done by deamination, insertion, or deletion of nucleotides.

Transcription in eukaryotes is a complex and highly regulated process that ensures accurate and timely expression of genes. It is essential for cellular function and differentiation.

Transcription is the process by which the information in a strand of DNA is copied into a new molecule of RNA. It is the first step of gene expression, in which a particular segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA polymerase. It results in a complementary, antiparallel RNA strand called a primary transcript.

Transcription occurs in eukaryotes in a way that is similar to prokaryotes with reference to the basic steps involved. However, some major differences between them include:

• Initiation is more complex. Eukaryotes have three types of RNA polymerases (I, II and III), each responsible for transcribing different types of RNA. Each RNA polymerase requires the assistance of several other proteins or protein complexes, called general (or basal) transcription factors, which must assemble into a complex on the promoter in order for RNA polymerase to bind and start transcription. Most promoters for RNA polymerase II also have a conserved sequence called the TATA box, which is recognized by a subunit of the transcription factor TFIID. Some genes lack a TATA box and have an initiator element instead.
• Termination does not involve stem-loop structures. Eukaryotic RNA polymerases do not terminate transcription at a specific site but rather transcription can stop at varying distances downstream of the gene. The transcript is cleaved at an internal site before RNA polymerase II finishes transcribing. This releases the upstream portion of the transcript, which will serve as the initial RNA prior to further processing (the pre-mRNA in the case of protein-encoding genes). The remainder of the transcript is digested by a 5′-exonuclease while it is still being transcribed by the RNA polymerase II. When the 5′-exonulease “catches up” to RNA polymerase II by digesting away all the overhanging RNA, it helps disengage the polymerase from its DNA template, finally terminating that round of transcription.
• Transcription is carried out by three enzymes (RNA polymerases I, II and III). Each of them transcribes different types of genes and has different subunits and properties. RNA polymerase I transcribes ribosomal RNA (rRNA) genes, except for 5S rRNA. RNA polymerase II transcribes protein-coding genes, resulting in messenger RNA (mRNA), and also some small nuclear RNAs (snRNAs) and microRNAs (miRNAs) involved in RNA processing and regulation. RNA polymerase III transcribes transfer RNA (tRNA) genes, 5S rRNA gene, and some other small RNAs.
• The regulation of transcription is more extensive than prokaryotes. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The accessibility of DNA to RNA polymerases and transcription factors depends on the chromatin state, which can be modified by various mechanisms such as histone modifications, chromatin remodeling, DNA methylation, and non-coding RNAs. Moreover, eukaryotic transcription can be regulated by enhancers and silencers, which are distant regulatory elements that can activate or repress transcription by interacting with promoters through DNA looping. Eukaryotic transcription can also be influenced by various signals from inside or outside the cell, such as hormones, growth factors, stress, and environmental cues.

Unlike prokaryotes, where a single RNA polymerase can synthesize all types of RNA, eukaryotes have three different RNA polymerases that are specialized for transcribing different classes of genes. These enzymes are named RNA polymerase I, II and III (or Pol I, II and III for short) and are located in the nucleus of the cell. Each RNA polymerase consists of multiple subunits, some of which are shared among the three enzymes and some of which are unique. The genes encoding some of the subunits show similarity to the bacterial RNA polymerase subunits, indicating a common evolutionary origin.

RNA polymerase I is responsible for transcribing most of the ribosomal RNA (rRNA) genes, which encode the structural and functional components of the ribosome. These genes are clustered in a specialized region of the chromosome called the nucleolus, where rRNA processing and ribosome assembly also take place. The rRNA genes transcribed by Pol I include the 28S, 18S and 5.8S rRNA genes, which together form the major rRNA molecule in eukaryotes.

RNA polymerase II is the main enzyme involved in transcribing protein-coding genes, which produce messenger RNA (mRNA) that is translated into polypeptides. Pol II also transcribes some non-coding RNAs that are involved in RNA processing and regulation, such as small nuclear RNAs (snRNAs) that form part of the spliceosome complex, and small nucleolar RNAs (snoRNAs) that modify rRNAs. One exception is the U6 snRNA gene, which is transcribed by Pol III.

RNA polymerase III transcribes the genes for transfer RNA (tRNA), which carry amino acids to the ribosome during translation. Pol III also transcribes some other small non-coding RNAs, such as 5S rRNA, which is part of the ribosome, U6 snRNA, which is part of the spliceosome, and 7S RNA, which is part of the signal recognition particle (SRP) that directs proteins to the endoplasmic reticulum membrane.

The three RNA polymerases have different promoter sequences that they recognize and bind to initiate transcription. They also require different sets of transcription factors to assemble on the promoter and recruit the enzyme. The transcription factors for Pol II are named TFIIA, TFIIB, TFIID and so on, while those for Pol I and Pol III are named TIF-IA, TIF-IB, TIF-ID and so on. The transcription factors help position the RNA polymerase correctly on the promoter, unwind the DNA template, phosphorylate the enzyme to start transcription, and regulate the rate and specificity of transcription.

Transcription in eukaryotes is the process of copying genetic information from DNA into RNA, especially mRNA, by the enzyme RNA polymerase. Eukaryotes have three types of RNA polymerase, each transcribing a different type of gene. Transcription in eukaryotes occurs in the nucleus and involves three stages: initiation, elongation, and termination. Some of the features that distinguish eukaryotic transcription from prokaryotic transcription are:

• Chromatin structure: Eukaryotic DNA is packaged into nucleosomes and higher order chromatin structures, which affect the accessibility of the DNA to RNA polymerase and transcription factors. Chromatin remodeling complexes and histone modifications can alter the chromatin structure and regulate transcription.
• Promoter complexity: Eukaryotic promoters are more diverse and complex than prokaryotic promoters. They can contain multiple elements that bind different transcription factors and regulate transcription in a tissue-specific, developmental, or environmental manner. The most common core promoter element in eukaryotes is the TATA box, which binds the TATA-binding protein (TBP), a subunit of the transcription factor TFIID. However, many eukaryotic genes lack a TATA box and have other core promoter elements, such as initiator (Inr), downstream promoter element (DPE), or TFIIB recognition element (BRE).
• Transcription initiation complex: Eukaryotic RNA polymerases cannot bind to the promoter directly, but require the assistance of several other proteins or protein complexes, called general (or basal) transcription factors. These transcription factors assemble into a complex on the promoter, forming a transcription pre-initiation complex (PIC), which recruits the appropriate RNA polymerase. For instance, RNA polymerase II requires at least six general transcription factors: TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. The PIC formation is also regulated by other proteins, such as activators and repressors, which bind to enhancer or silencer sequences and modulate transcription rate.
• RNA processing: Eukaryotic transcripts undergo various processing steps before they become functional RNAs. These include 5` capping, 3` polyadenylation, splicing, and nucleotide modifications. 5` capping involves the addition of a 7-methylguanosine cap to the 5` end of the transcript, which protects it from degradation and facilitates translation initiation. 3` polyadenylation involves the cleavage of the transcript at a specific site and the addition of a poly(A) tail to the 3` end, which stabilizes the transcript and enhances translation efficiency. Splicing involves the removal of non-coding introns and the joining of coding exons by a complex called spliceosome. Nucleotide modifications involve chemical changes to some nucleotides in the transcript, such as methylation or deamination, which can affect the structure and function of the RNA.
• RNA transport: Eukaryotic transcripts are synthesized in the nucleus but need to be transported to the cytoplasm for translation. The transport of RNAs across the nuclear envelope is mediated by nuclear pore complexes and specific export factors that recognize different types of RNAs. For instance, mRNA export requires a protein complex called TREX (transcription-export complex), which binds to mRNA during transcription and recruits other export factors that interact with nuclear pore complexes.

These features make eukaryotic transcription a tightly regulated process that requires a variety of proteins to interact with each other and with the DNA strand. Eukaryotic transcription allows for precise control of gene expression in response to various signals and conditions.

The basic mechanism of RNA synthesis by these eukaryotic RNA polymerases can be divided into the following phases:

Initiation Phase

During initiation, RNA polymerase recognizes a specific site on the DNA, upstream from the gene that will be transcribed, called a promoter site and then unwinds the DNA locally . Most promoter sites for RNA polymerase II include a highly conserved sequence located about 25–35 bp upstream (i.e. to the 5 side) of the start site which has the consensus TATA(A/T)A(A/T) and is called the TATA box . Since the start site is denoted as position +1, the TATA box position is said to be located at about position -25 . The TATA box sequence resembles the -10 sequence in prokaryotes (TATAAT) except that it is located further upstream . Both elements have essentially the same function, namely recognition by the RNA polymerase in order to position the enzyme at the correct location to initiate transcription . The sequence around the TATA box is also important in that it influences the efficiency of initiation . Transcription is also regulated by upstream control elements that lie 5′ to the TATA box .

Some eukaryotic protein-coding genes lack a TATA box and have an initiator element instead, centered around the transcriptional initiation site .

In order to initiate transcription, RNA polymerase II requires the assistance of several other proteins or protein complexes, called general (or basal) transcription factors, which must assemble into a complex on the promoter in order for RNA polymerase to bind and start transcription . These all have the generic name of TFII (for Transcription Factor for RNA polymerase II) .

The first event in initiation is the binding of the transcription factor IID (TFIID) protein complex to the TATA box via one its subunits called TBP (TATA box binding protein) . As soon as the TFIID complex has bound, TFIIA binds and stabilizes the TFIID-TATA box interaction . Next, TFIIB binds to TFIID . However, TFIIB can also bind to RNA polymerase II and so acts as a bridging protein . Thus, RNA polymerase II, which has already complexed with TFIIF, now binds . This is followed by the binding of TFIIE and H . This final protein complex contains at least 40 polypeptides and is called the transcription initiation complex .

Those protein-coding genes that have an initiator element instead of a TATA box appear to need another protein(s) that binds to the initiator element . The other transcription factors then bind to form the transcription initiation complex in a similar manner to that described above for genes possessing a TATA box promoter .

Elongation Phase

TFIIH has two functions:

• It is a helicase, which means that it can use ATP to unwind the DNA helix, allowing transcription to begin.
• In addition, it phosphorylates RNA polymerase II which causes this enzyme to change its conformation and dissociate from other proteins in the initiation complex.

The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain (CTD) of the RNA polymerase II molecule. Interestingly, only RNA polymerase II that has a non-phosphorylated CTD can initiate transcription but only an RNA polymerase II with a phosphorylated CTD can elongate RNA. RNA polymerase II now starts moving along the DNA template, synthesizing RNA, that is, the process enters the elongation phase. RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on an incoming ribonucleoside 5-triphosphate. The RNA molecule made from a protein-coding gene by RNA polymerase II is called a primary transcript.

Termination Phase

Elongation of the RNA chain continues until termination occurs. Unlike RNA polymerase in prokaryotes, RNA polymerase II does not terminate transcription at a specific site but rather transcription can stop at varying distances downstream of the gene. RNA genes transcribed by RNA Polymerse II lack any specific signals or sequences that direct RNA Polymerase II to terminate at specific locations. RNA Polymerase II can continue to transcribe RNA anywhere from a few bp to thousands of bp past the actual end of the gene. The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing. This releases the upstream portion of the transcript, which will serve as the initial RNA prior to further processing (the pre-mRNA in the case of protein-encoding genes.) This cleavage site is considered the “end” of the gene. The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the RNA Polymerase II. When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the overhanging RNA, it helps disengage the polymerase from its DNA template strand, finally terminating that round of transcription.

The primary eukaryotic mRNA transcript is much longer and localized into the nucleus, when it is also called heterogeneous nuclear RNA (hnRNA) or pre-mRNA. It undergoes various processing steps to change into a mature RNA that can be transported to the cytoplasm and translated by the ribosomes. These steps include:

• Cleavage: Larger RNA precursors are cleaved to form smaller RNAs. For instance, the primary transcript of a protein-coding gene is cleaved by an endonuclease at a specific site downstream of the coding region, releasing the upstream portion of the transcript, which will serve as the initial RNA prior to further processing (the pre-mRNA). The remainder of the transcript is degraded by a 5`-exonuclease.
• Capping and Tailing: At the 5` end, a cap (consisting of 7-methyl guanosine or 7 mG) is added by a capping enzyme. The cap protects the RNA from degradation by exonucleases and also facilitates its recognition by the ribosome during translation. At the 3` end, a tail of poly A (a string of adenine nucleotides) is added by a polyadenylate polymerase. The poly-A tail also protects the RNA from degradation and helps in its export from the nucleus .
• Splicing: The eukaryotic primary mRNAs are made up of two types of segments: non-coding introns and the coding exons. The introns are removed by a process called RNA splicing, where a complex of proteins and small RNAs called the spliceosome recognizes specific sequences at the boundaries of introns and exons and catalyzes their excision and ligation. Splicing allows the removal of non-functional or unwanted sequences from the mRNA and also enables alternative splicing, which is the production of different mature mRNA molecules from the same initial transcript by using different combinations of exons .
• Nucleotide Modifications: Some nucleotides in the RNA are chemically modified after transcription to alter their structure or function. These modifications are most common in tRNA and rRNA, but can also occur in mRNA. Some examples of nucleotide modifications are methylation (e.g., methyl cytosine, methyl guanosine), deamination (e.g., inosine from adenine), reduction (e.g., dihydrouracil), and pseudouridylation (e.g., pseudouracil).

Post-transcription processing is required to convert primary transcript into functional RNAs that can participate in protein synthesis or perform other cellular roles.

Transcription is the process by which the information in a strand of DNA is copied into a new molecule of RNA. It is the first step of gene expression, in which a particular segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA polymerase. Transcription has several important functions and implications for cells and organisms:

• Transcription maintains the link between DNA and RNA, allowing cells to use a stable nucleic acid as the genetic material while retaining most of their protein synthesis machinery.
• Transcription enables the regulation of gene expression, as different genes can be transcribed at different times or in different tissues depending on the need for their products. Transcription is controlled by various factors that bind to promoter regions and other regulatory elements on DNA.
• Transcription allows for the generation of diversity and complexity in eukaryotes, as the primary transcripts can undergo various processing steps such as splicing, capping, and tailing before becoming mature RNAs. These processes can produce different variants of RNAs from the same gene, which can have different functions or interactions.
• Transcription is essential for the synthesis of proteins, which are the main functional and structural molecules of cells and organisms. Proteins are produced by translating the mRNA transcripts into amino acid chains by the ribosomes. Proteins perform various roles such as catalyzing reactions, transporting molecules, signaling pathways, and forming structures.

Transcription is therefore a vital process that enables life to exist and evolve. Without transcription, cells would not be able to express their genes or produce their proteins, which would result in loss of function and death.

We are Compiling this Section. Thanks for your understanding.