Eukaryotic Transcription: Key Stages, Regulatory Mechanisms, Products, and Significance

Eukaryotic transcription is a key initial step of gene expression, which occupies a core position in the central rule. It uses DNA as a template to synthesize various RNA molecules by RNA polymerase. Its regulatory network is fine and complex, and it is widely involved in life activities such as embryonic development, cell differentiation, and tissue homeostasis maintenance.
In recent years, high-throughput technological innovations such as ChIP-seq and scRNA-seq have greatly promoted the study of transcription regulation mechanisms and revealed many key regulatory factors and modes of action. However, the dynamic assembly law of transcription initiation complex, the synergistic mechanism of transcription extension and chromatin remodeling, and the relationship between transcription abnormality and major diseases still need further exploration. Clarifying these mechanisms is of far-reaching significance for understanding the essence of life and developing targeted therapy.
This paper systematically introduces the key stages, regulation mechanism, transcription products and their functions of eukaryotic transcription, and expounds its core position and its important significance in disease research.

What is Eukaryotic Transcription

Eukaryotic transcription refers to synthesizing RNA according to the principle of base complementary pairing in eukaryotic cells with a strand of DNA as a template and with the participation of various protein factors such as RNA polymerase. This process is the initial link of gene expression and plays a key role in genetic information transmission. In transcription, three kinds of RNA polymerases (RNA polymerases I, II, and III) perform their respective duties.

• RNA Polymerase I exclusively synthesizes ribosomal RNA (rRNA) precursors, essential for ribosome assembly—the sites of protein production.
• RNA Polymerase II transcribes protein-coding genes, partnering with general transcription factors (e.g., TFIID, TFIIB) at promoter regions like the TATA box to initiate transcription.
• RNA Polymerase III generates small RNAs, including transfer RNA (tRNA) and 5S rRNA.

This spatial isolation not only ensures that the transcription process is free from the interference of the complex metabolic environment in the cytoplasm, but also precisely regulates the transportation of transcription products through the nuclear pore complex, ensuring that mature RNA molecules can function at the right time and place, thus laying a stable and orderly foundation for the subsequent synthesis of protein and maintaining the normal life activities of cells.

Schematic representation of the different mechanisms of transposition (Colonna et al., 2022)

Three Main Period in Eukaryotic Transcription

Eukaryotic transcription can be divided into three closely connected stages: initiation, extension, and termination. Each stage is dominated by a specific molecular mechanism; each cooperates with the other, which ensures the accurate transcription of genetic information and lays the foundation for subsequent life activities.

Initial stage

The initial stage is the key step of transcription, which requires RNA polymerase to combine with a specific region-promoter on the DNA template. There are three different types of RNA polymerases in eukaryotes, which are responsible for the transcription of genes. Take RNA polymerase II as an example. It first assembles with common transcription factors to form a pre-initiation complex (PIC), and then recognizes and binds to cis-acting elements such as the TATA box in the promoter region. With the help of other transcription activating factors, it opens the DNA double-strand and exposes the template chain, creating conditions for RNA synthesis.

Cryo-electron microscopy (EM) analysis of promoter-bound TFIID (Nogales et al., 2017)

Extension stage

When the initial complex forms and begins to synthesize RNA chains, transcription enters the extension stage. RNA polymerase moves along the 3’→5′ direction of the DNA template chain. Four kinds of ribonucleotides (NTP) are used as raw materials, and according to the principle of base complementary pairing (A-U, T-A, G-C, C-G), nucleotides are added to the 3′-OH end of the new RNA chain in turn, so that the RNA chain continuously extends from the 5′ end to the 3′ end.
In this process, RNA polymerase has the dual functions of unwinding and polymerization. It unties the front DNA double strand and synthesizes the RNA strand, and at the same time, the transcribed DNA double strand is restored to the double helix structure. In addition, some extension factors are needed in the extension process to ensure efficient and accurate transcription.

Termination stage

The termination process of transcription is complicated, and the termination mechanism of different types of RNA polymerases is different.

• For RNA polymerase II, transcription termination is usually closely related to post-transcriptional processing. When RNA polymerase II is transcribed into the termination signal region of the gene, it will trigger a series of events. First, the 3′ end of the transcription product RNA will be cut, and then a poly-A tail will be added under the action of poly-A polymerase. At the same time, RNA polymerase II continued to transcribe for a certain distance, and finally detached from the DNA template to complete the transcription termination process.
• The termination mechanism of RNA polymerases I and III is relatively simple. They dissociate RNA polymerases from the template by recognizing specific DNA termination sequences, thus ending transcription.

Eukaryotic transcription cycle (Rodríguez-Molina et al., 2023)

Eukaryotic Transcription Regulation Mechanism

Eukaryotic transcription regulation is a complex process with multi-level and multi-factor cooperative participation. Through the precise interaction between cis-acting elements and trans-acting factors, the dynamic changes of chromatin structure and the diverse regulation of non-coding RNA, the gene can be properly expressed in appropriate time and space to maintain the normal function of cells and the steady state of organisms.

Cis-acting Element

Cis-acting elements refer to specific nucleotide sequences on DNA molecules related to gene transcription regulation. They are usually located in the upstream and downstream regions of genes and can only affect gene expression on the same DNA chain, mainly including the following types:

• Promoter: A DNA sequence located upstream of the transcription initiation site of a gene, which can specifically bind with RNA polymerase and other transcription factors to start gene transcription, and is a key element of gene expression regulation.
• Enhancer: It can enhance the activity of the promoter and improve the transcription efficiency of the gene. Its function has nothing to do with position and direction, and it can be located in the upstream, downstream, or intron of a gene.
• Silencer: Contrary to enhancer, it can inhibit gene transcription and reduce the gene expression level.

Structure of RNA polymerase II-NCP complex (Farnung et al., 2018)

Trans-acting Element

Trans-acting factors refer to protein factors that can directly or indirectly interact with cis-acting elements and regulate gene transcription, also known as transcription factors. They usually have the following domains.

• DNA binding domain: It can specifically recognize and bind specific DNA sequences in cis-acting elements.
• Transcription activation domain: It can interact with other transcription factors or RNA polymerase to promote the formation of the transcription initiation complex and activate gene transcription.
• Dimerization domain: Some trans-acting factors enhance the binding ability and specificity with DNA by forming dimers.

Trans-acting factors finely regulate the transcription of eukaryotic genes through their precise combination with cis-acting elements and their interaction genes can be accurately expressed in different cell types, development stages, and environmental conditions to meet the needs of organism growth, development, and adaptation to the environment.

Chromatin Structure Regulation

Eukaryotic DNA combines with histone to form chromatin, and the structural state of chromatin has an important influence on transcription. Chromatin can undergo structural changes under the action of histone modification (such as methylation, acetylation, phosphorylation, etc.) and an ATP-dependent chromatin remodeling complex.

• Histone acetylation will loosen chromatin structure, which is beneficial to the combination of transcription factors and RNA polymerase with DNA and promote transcription.
• Histone methylation may promote or inhibit transcription according to the different modification sites and degrees.
• The chromatin remodeling complex can regulate chromatin accessibility by changing the position and composition of nucleosomes on DNA, and then regulate the transcription process.

The nucleosome contains specific interactions that provide a barrier to transcript elongation and can be disassembled through chromatin remodelers (Petesch et al., 2012)

Non-coding RNA Regulation

In recent years, it has been found that non-coding RNA (such as miRNA and lncRNA) plays an important role in eukaryotic transcription regulation. MiRNA can inhibit the translation or promote the degradation of mRNA through complementary pairing with the mRNA of the target gene, which indirectly affects the regulation of gene expression at the post-transcription level. At the same time, some miRNAs can also affect the transcription process through interaction with transcription factors. LncRNA can regulate gene expression at the transcription level and post-transcription level by interacting with DNA, RNA, or protein.

Eukaryotic Transcription Production and Their Function

Transcription products of eukaryotes include messenger RNA, transporter RNA, ribosomal RNA, which is the key component of ribosomes, and all kinds of non-coding RNA that play a regulatory role. They work together to maintain the normal operation of cells and the orderly progress of life activities.

mRNA

MRNA is the most important kind of transcription product, which carries the genetic information transcribed from DNA and serves as a template for protein synthesis. In eukaryotes, the newly synthesized mRNA precursor (hnRNA) needs complicated processing, including capping at the 5′ end (adding 7-methylguanosine triphosphate cap), tailing at the 3′ end (adding poly-A tail), and splicing (removing introns and connecting exons) to form mature mRNA.
Mature mRNA enters the cytoplasm through the nuclear pore and combines with ribosomes. With the help of Trna and various translation factors, amino acids are connected in turn to form polypeptide chains according to the codon sequence on mRNA, and finally folded into a protein with a specific spatial structure and biological function.

tRNA

TRNA is responsible for transporting amino acids during the synthesis of protein. The TRNA molecule is small, containing about 70-90 nucleotides, and its secondary structure is clover-shaped, with an amino acid arm, anticodon loop, and other structures. The amino acid arm can specifically bind the corresponding amino acid, and the anti-codon on the anticodon loop can complement and pair with the codon on the mRNA, thus ensuring that the amino acid is accurately incorporated into the polypeptide chain according to the codon order of the mRNA. TRNA is transcribed from RNA polymerase III. After transcription, it also needs a series of modification processes, such as the formation of rare bases and the addition of a 3′-terminal CCA sequence, to form an active tRNA molecule.

RNase P cleaves the 5′ leader from precursor tRNAs (Walker et al., 2006)

rRNA

rRNA is an important component of ribosomes, and participates in the site construction of protein synthesis. There are four kinds of rRNA in eukaryotes, which are 28S rRNA, 18S rRNA, 5.8S rRNA, and 5S rRNA. Among them, 28S rRNA, 18S rRNA, and 5.8S rRNA are transcribed by RNA polymerase I, and they are processed after transcription in the nucleolus and assembled with various ribosomal proteins to form large and small subunits of ribosomes. 5S rRNA is transcribed by RNA polymerase III, and then enters the nucleolus to participate in ribosome assembly. In the process of protein synthesis, ribosomes combine with mRNA to provide a place for the combination of amino acids carried by tRNA and the synthesis of polypeptide chains, and play the role of catalysis and scaffold.

Other non-coding RNA

In addition to the above three main RNAs, eukaryotic transcription will also produce a variety of non-coding RNAs, such as small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and so on. snRNAs are mainly involved in the splicing process of mRNA precursors. They combine with protein to form small nuclear ribonucleoprotein particles (snRNP), which help to remove introns and connect exons by recognizing and combining the splicing sites on mRNA precursors. snoRNA is mainly involved in the modification process of rRNA and tRNA, such as guiding the methylation and pseudo-uracil modification of rRNA to ensure the normal structure and function of rRNA and tRNA.

Conclusion

Eukaryotic transcription, as the initial key link of gene expression, has formed a precise life regulation network from the complex initiation, extension and termination process, to the multi-level cis-elements and trans-factors, chromatin structure and non-coding RNA regulation mechanism, and then to the diverse products such as mRNA, tRNA and rRNA performing different functions. With the rise of high-throughput sequencing technology, RNA-seq has achieved in-depth analysis of transcripts, ATAC-seq and ChIP-seq have helped to analyze the molecular mechanism of transcription regulation, and scRNA-seq has unlocked the mystery of transcription heterogeneity at the cellular level.
These technological innovations have greatly promoted the research process of eukaryotic transcription. Looking forward to the future, cutting-edge technologies such as nanopore sequencing and spatial transcriptome sequencing are expected to achieve more detailed transcription research on time and space scales, which will help us further uncover the mystery of life activities and open up new paths for disease diagnosis and treatment, bioengineering, and other fields.

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