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8.3: Eukaryotic Transcription - Biology

8.3: Eukaryotic Transcription - Biology


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Transcription in eukaryotes is more complicated, but follows the same general ideas. It also makes other untranslated RNAs such as tRNAs and a variety of small nuclear RNAs. All of the eukaryotic RNA polymerases are composed of two large subunits, roughly analogous to the β and β’ subunits of prokaryotic RNAP, but instead of just three or four other subunits, there are over a dozen smaller subunits to the eukaryotic RNA polymerase holoenzymes.

Initiation of transcription is also much more complicated. Not only is there great variety in promoters recognized by RNAP II, both RNAP I and RNAP III recognize promoters with particular structural characteristics.

The eukaryotic RNA polymerases were named I, II, and III based on their elution order from ion-exchange chromatography purification. They are also partially distinguishable by their sensitivity to α-amanitin and related amatoxin-family mushroom poisons. RNAP I (and prokaryotic RNAP) is insensitive to these toxins, RNAP III is somewhat sensitive (Kd ~10-6 M), and RNAP II is highly sensitive (Kd ~10-8 M). These toxins act by binding to a site in the RNA-DNA cleft and interfering with translocation of the RNA. That is, there is no problem with importing a nucleotide or with attaching it to the new RNA, but the RNA strand cannot move through the active site and allow the next nucleotide to be added.

One of the most common eukaryotic RNAP II promoters is the TATA box, named for the highly conserved motif that defines it. Although it appears similar to the Pribnow box in prokaryotes, it is generally located further upstream from the start site, and its position is far more variable. Whereas the Pribnow box is located at -10, the TATA box may be located closer to -30 +/- 4. Also, rather than just a sigma factor to recognize the promoter in conjunction with the polymerase core enzyme, the eukaryotic promoter is recognized by a multi-subunit complex called transcription factor IID (TFIID). TFIID is comprised of TATA-binding protein (TBP) and several TBP-associated factors (TAFs).

This binding of the promoter by TFIID occurs independently of RNA Polymerase II, and in fact, RNAP II will not attach to TFIID at this time. After TFIID has bound the TATA box, two more transcription factors, TFIIA and TFIIB, attach to the TFIID as well as the nearby DNA, stabilizing the complex. TFIIF attaches to TFIID and TFIIB to allow docking of the RNA Polymerase II. The complex is still not ready to begin transcription: two more factors are required. TFIIE binds TFIIF and RNAP II, and finally, TFIIH attaches to RNAP II, providing a helicase activity needed to pry apart the two strands of DNA and allow the polymerase to read one of them. TFIIH also has another important enzymatic activity: it is also a serine kinase that phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II. There are several serines in the CTD, and as they are sequentially phosphorylated, the CTD extends like a (negatively charged) tail and helps to promote separation between the RNAP II and the TFIID/promoter.

Elongation of the RNA strand in eukaryotes is very similar to that in prokaryotes with the obvious difference that transcription occurs in the nucleus rather than in the cytoplasm. Thus, in prokaryotes, the RNA can be used for translation of proteins even as it is still being transcribed from the DNA! In eukaryotes, the situation is significantly more complex: there are a number of post-transcriptional events (5’ end-capping, 3’ polyadenylation, and often RNA splicing) that must occur before the RNA is ready to be transported out of the nucleus and made available for translation in the cytoplasm.

Termination of eukaryotic transcription is not well-described at this writing. RNAP I appears to require a DNA-binding termination factor, which is not analogous to the prokaryotic Rho factor, which is an RNA binding protein. RNAP III terminates transcription without any external factor, and this termination usually occurs after adding a series of uridine residues. However, it does not appear to use the hairpin loop structure found in rho-independent bacterial transcription. The termination of protein-coding RNAP II transcripts is linked to an enzyme complex that also cleaves part of the 3’ end of the RNA off, and adds a poly-A tail. However, it is not clear how the polyadenylation complex is involved in determining the point of transcription termination, which can be over 1000 nucleotides beyond the poly-A site (e.g. the β-globin gene in Mus musculus). Upon termination and release from the RNAP II and template DNA, the RNA is known as the primary transcript, but must undergo post-transcriptional processing before it is a mature messenger RNA (mRNA) ready to be exported to the cytoplasm and used to direct translation.


Transcription in Eukaryotes | Genetics

Transcription has been defined in various ways. Some definitions of transcription are given here. The synthesis of RNA from a single strand of a DNA molecule in the presence of enzyme RNA polymerase is called transcription. In other words, the process of formation of a messenger RNA molecule using a DNA molecule as a template is referred to as transcription.

The main points related to transcription in eukaryotes are briefly discussed below:

RNA is synthesized from a DNA template. The RNA is processed into messenger RNA [mRNA], which is then used for synthesis of a protein. The RNA thus synthesized is called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein- synthesizing machinery of the cell.

The main difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the T, of thymine of DNA.

The RNA is synthesized from a single strand or template of a DNA molecule. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A transcription unit codes the sequence that is translated into protein. It also directs and regulates protein synthesis.

The DNA strand which is used in RNA synthesis is called template strand because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The DNA strand which does not take part in DNA synthesis is called coding strand, because, its nucleotide sequence is the same as that of the newly created RNA transcript.

The process of transcription is catalyzed by the specific enzyme called RNA polymerase. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand. In eukaryotes, there are three classes of RNA polymerases: I, II and III which are involved in the transcription of all protein genes.

4. Genetic Information Copied:

In this process, the genetic information coded in DNA is copied into a molecule of RNA. The genetic information is transcribed or copied, from DNA to RNA. In other words, it results in the transfer of genetic information from DNA into RNA.

The expression of a gene consists of two major steps, viz., transcription and translation. Thus transcription is the first step in the process of gene regulation or protein synthesis.

6. Direction of Synthesis:

As in DNA replication, RNA is synthesized in the 5′ —> 3′ direction. The DNA template strand is read 3′ –> 5′ by RNA polymerase and the new RNA strand is synthesized in the 5′ -> 3′ direction. RNA polymerase binds to the 3′ end of a gene (promoter) on the DNA template strand and travels toward the 5′ end.

The regulatory sequence that is before, or 5′, of the coding sequence is called 5′ un-translated region (5′ UTR), and sequence found following, or 3′, of the coding sequence is called 3′ un-translated region (3′ UTR). Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA therefore, transcription has a lower copying fidelity than DNA replication.

Mechanism of Transcription in Eukaryotes:

The mechanism of transcription consists of five major steps, viz:

These are briefly discussed as follows:

1. Pre-Initiation:

The initiation of transcription does not require a primer to start. RNA polymerase simply binds to the DNA and, along with other cofactors, unwinds the DNA to create an initiation bubble so that the RNA polymerase has access to the single-stranded DNA template. However, RNA Polymerase does require a promoter like sequence.

Proximal (core) Promoters:

TATA promoters are found around -30 bp to the start site of transcription. Not all genes have TATA box promoters and there exists TATA-less promoters as well. The TATA promoter consensus sequence is TATA(A/T)A(A/T).

In eukaryotes and archaea, transcription initiation is far more complex. The main difference is that eukaryotic polymerases do not recognize directly their core promoter sequences. In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription.

Only after attachment of certain transcription factors to the promoter, the RNA polymerase binds to it. The complete assembly of transcription factors and RNA polymerase bind-to the promoter, called transcription initiation complex. Initiation starts as soon as the complex is opened and the first phosphodiester bond is formed. This is the end of Initiation.

RNA Pol II does not contain a subunit similar to the prokaryotic factor, which can recognize the promoter and unwind the DNA double helix. In eukaryotes, these two functions are carried out by a set of proteins called general transcription factors.

The RNA Pol II is associated with six general transcription factors, designated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, where “TF” stands for “transcription factor” and “II” for the RNA Pol II.

TFIID consists of TBP (TATA-box binding protein) and TAFs (TBP associated factors). The role of TBP is to bind the core promoter. TAFs may assist TBP in this process. In human cells, TAFs are formed by 12 subunits. One of them, TAF250 (with molecular weight 250 kD), has the histone acetyltransferase activity, which can relieve the binding between DNA and histones in the nucleosome.

The transcription factor which catalyzes DNA melting is TFIIH. However, before TFIIH can unwind DNA, the RNA Pol II and at least five general transcription factors (TFIIA is not absolutely necessary) have to form a pre-initiation complex (PIC).

3. Promoter Clearance:

After the first bond is synthesized the RNA polymerase must clear the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both Eukaryotes and Prokaryotes.

Once the transcript reaches approximately 23 nucleotides it no longer slips and elongation can occur. This is an ATP dependent process. Promoter clearance also coincides with Phosphorylation of serine 5 on the carboxy terminal domain which is phosphorylated by TFIIH.

For RNA synthesis, one strand of DNA known as the template strand or non-coding strand is used as a template. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy.

Although RNA polymeras traverses the template strand from 3′ —> 5′, the coding (non-template) strand is usually used as the reference point, so transcription is said to go from 5′ —> 3′.

This produces an RNA molecule from 5′ —> 3′, an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).

After pre-initiation complex [PIC] is assembled at the promoter, TFIIH can use its helicase activity to unwind DNA. This requires energy released from ATP hydrolysis. The DNA melting starts from about -10 bp.

Then, RNA Pol II uses nucleoside triphosphates (NTPs) to synthesize a RNA transcript. During RNA elongation, TFIIF remains attached to the RNA polymerase, but all of the other transcription factors have dissociated from PIC.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II is critical for elongation. In the initiation phase, CTD is un-phosphorylated, but during elongation it has to be phosphorylated. This domain contains many proline, serine and threonine residues.

In eukaryotic transcription the mechanism of termination is not very clear. In other words, it is not well understood. It involves cleavage of the new transcript, followed by template- independent addition of As at its new 3′ end, in a process called polyadenylation.

Eukaryotic protein genes contain a poIy-A signal located downstream of the last exon. This signal is used to add a series of adenylate residues during RNA processing. Transcription often terminates at 0.5-2 kb downstream of the poly-A signal.

Transcription Factories in Eukaryotes:

Active transcription units that are clustered in the nucleus, in discrete sites are called ‘transcription factories’. Such sites could be visualized after allowing, engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labelling the tagged nascent RNA.

Transcription factories can also be localized using fluorescence in situ hybridization, or marked by antibodies directed against polymerases. There are

10,000 factories in the nucleoplasm of a HeLa cell, among which are

8,000 polymerase II factories and

2,000 polymerase III factories. Each polymerase II factory contains

As most active transcription units are associated with only one polymerase, each factory will be associated with

8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factory.

Reverse Transcription in Eukaryotes:

Synthesis of DNA from RNA molecule in the presence of enzyme reverse transcriptase is referred to as reverse transcription. Reverse transcription was first reported by Temin and Baltimore in 1970 for which they were awarded Nobel prize in 1975. Reverse transcription is also known as Teminism. Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.

In some eukaryotic cells, an enzyme is found with reverse transcription activity. It is called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes DNA repeating sequence, or “junk” DNA. This repeated sequence of “junk” DNA is important because every time a linear chromosome is duplicated, it is shortened in length.

With “junk” DNA at the ends of chromosomes, the shortening eliminates some repeated, or junk sequence, rather than the protein-encoding DNA sequence that is further away from the chromosome ends.

Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes without losing important protein-coding DNA sequence. Activation of telomerase can be part of the process that allows cancer cells to become immortal.

Role of Transcription Factors in Eukaryotes:

In eukaryotes, the association between DNA and histones prevents access of the polymerase and general transcription factors to the promoter. Histone acetylation catalyzed by HATs can relieve the binding between DNA and histones. Although a subunit of TFIID (TAF250 in human) has the HAT activity, participation of other HATs can make transcription more efficient. The following rules apply to most (but not all).

(i) Binding of activators to the enhancer element recruits HATs to relieve association between histones and DNA, thereby enhancing transcription.

(ii) Binding of repressors to the silencer element recruits histone deacetylases (denoted by HDs or HDACs) to tighten association between histones and DNA.


Initiation

The eukaryotic promoters that we are most interested in are similar to prokaryotic promoters in that they contain a TATA box (Figure 1). However, initiation of transcription is much more complex in eukaryotes compared to prokaryotes. Unlike the prokaryotic RNA polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase.

Figure 1 The generalized structure of a eukaryotic promoter and transcription factors.

In addition, there are three different RNA polymerases in eukaryotes, each of which is made up of 10 subunits or more. Each eukaryotic RNA polymerase also requires a distinct set of transcription factors to bring it to the DNA template.

RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes. The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation. RNA polymerase I synthesizes most of the rRNAs.

RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. For clarity, the term “mRNA” will only be used to describe the mature, processed molecules that are ready to be translated. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes.

RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs including transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. The tRNAs have a critical role in translation they serve as the adaptor molecules between the mRNA template and the growing polypeptide chain. Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors.

Each of the types of RNA polymerase recognizes a different promoter sequence and requires different transcription factors.


Eukaryotic mRNA

Credit: Kelvinsong (CC-BY-3.0)
Eukaryotic genes may often contain introns (non-coding sequences) that are spliced out from the exons (coding sequences). This complexity permits for increased variety of gene products. Mature eukaryotic mRNAs conatins a 5′-methyl-Guanine followed by an untranslated leader sequence ( 5′-UTR ), the coding sequences ( cds ), a 3′-untranslated region ( 3′-UTR ) and a long stretch of Adenines (polyA tail).


Expression is most easily measured with RNA since nucleic acid manipulation is fairly simple with 4 different nucleotides. In eukaryotes, the messenger RNA (mRNA) intermediate that is transcribed from DNA contains a polyA tail that is used to separate these messages from other types of RNA that are abundant within cells (like ribosomal RNA). Through the use of an enzyme called reverse transcriptase (RT) and primers composed of deoxy-Thymidines ( oligo-dT or dT18), mRNA can be converted into a single strand of DNA that is complimentary to the mRNA. This complimentary DNA is called cDNA . cDNA is very stable compared to the highly labile mRNA and is used for subsequent processing.

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Section Summary

Transcription in eukaryotes involves one of three types of polymerases, depending on the gene being transcribed. RNA polymerase II transcribes all of the protein-coding genes, whereas RNA polymerase I transcribes rRNA genes, and RNA polymerase III transcribes rRNA, tRNA, and small nuclear RNA genes. The initiation of transcription in eukaryotes involves the binding of several transcription factors to complex promoter sequences that are usually located upstream of the gene being copied. The mRNA is synthesized in the 5′ to 3′ direction, and the FACT complex moves and reassembles nucleosomes as the polymerase passes by. Whereas RNA polymerases I and III terminate transcription by protein- or RNA hairpin-dependent methods, RNA polymerase II transcribes for 1,000 or more nucleotides beyond the gene template and cleaves the excess during pre-mRNA processing.

Additional Self Check Question

1. A scientist splices a eukaryotic promoter in front of a bacterial gene and inserts the gene in a bacterial chromosome. Would you expect the bacteria to transcribe the gene?


Promoter Structures for RNA Polymerases I and III

In eukaryotes, the conserved promoter elements differ for genes transcribed by RNA polymerases I, II, and III. RNA polymerase I transcribes genes that have two GC-rich promoter sequences in the -45 to +20 region. These sequences alone are sufficient for transcription initiation to occur, but promoters with additional sequences in the region from -180 to -105 upstream of the initiation site will further enhance initiation. Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves.


Eukaryotic Elongation and Termination

Following the formation of the preinitiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed as it does in prokaryotes with the polymerase synthesizing pre-mRNA in the 5' to 3' direction. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so this section will focus on how this polymerase accomplishes elongation and termination.

Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound around eight histones like thread around a spool.

For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. This is accomplished by a special protein complex called FACT , which stands for “facilitates chromatin transcription.” This complex pulls histones away from the DNA template as the polymerase moves along it. Once the pre-mRNA is synthesized, the FACT complex replaces the histones to recreate the nucleosomes.

The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.


Eukaryotic transcription

Transcription in eukaryotes: A brief view
Transcription is the process by which single stranded RNA is synthesized by double stranded DNA. Transcription in eukaryotes and prokaryotes has many similarities while at the same time both showing their individual characteristics due to the differences in organization. RNA Polymerase (RNAP or RNA Pol) is different in prokaryotes and eukaryotes. Coupled transcription is seen in prokaryotes but not in Eukaryotes. In eukaryotes the pre-RNA should be spliced first to be translated.
In Eukaryotic transcription, synthesis of RNA occurs in the 3’→5’ direction. The 3’ end is more reactive due to the hydroxide group. 5’ end containing phosphate groups meanwhile, is not very reactive when it comes to adding new nucleotides. In Eukaryotes, the whole genome is not transcribed at once. Only a part of the genome is transcribed which also acts as the first, principle stage of genetic regulation.
Eukaryotes have five nuclear polymerases:
• RNA Polymerase I: This produces rRNA (23S, 5.8S, and 18S) which are the major components in a ribosome. This also produces pre-rRNA in yeasts.
• RNA Polymerase II: Helps in the production of mRNA (messenger RNA), snRNA (small, nuclear RNA), miRNA. This is the most studied type and requires several transcription factors for its binding
• RNA Polymerase III: This synthesizes tRNA (transfer RNA), 5S rRNA and other small RNAs required in the cytosol and nucleus.
• RNA Polymerase IV: Synthesizes siRNA (small interfering RNA) in plants.
• RNA Polymerase V: This is the least studied polymerase and synthesizes siRNA-directed heterochromatin in plants.
Eukaryotic transcription can be broadly divided into 4 stages:
• Pre-Initiation
• Initiation
• Elongation
• Termination
Transcription is an elaborate process which cells use to copy the genetic information stored in DNA into RNA. This pre-RNA is modified into mRNA before being transcribed to proteins. Transcription is the first step to utilizing the genetic information in a cell. Both Eukaryotes and Prokaryotes employ this process with the basic phases remaining the same. However eukaryotic transcription is more complex indicating the changes transcription has undergone towards perfection during evolution.


Eukaryotic mRNA

Credit: Kelvinsong (CC-BY-3.0)
Eukaryotic genes may often contain introns (non-coding sequences) that are spliced out from the exons (coding sequences). This complexity permits for increased variety of gene products. Mature eukaryotic mRNAs conatins a 5&prime-methyl-Guanine followed by an untranslated leader sequence ( 5&prime-UTR ), the coding sequences ( cds ), a 3&prime-untranslated region ( 3&prime-UTR ) and a long stretch of Adenines (polyA tail).


Expression is most easily measured with RNA since nucleic acid manipulation is fairly simple with 4 different nucleotides. In eukaryotes, the messenger RNA (mRNA) intermediate that is transcribed from DNA contains a polyA tail that is used to separate these messages from other types of RNA that are abundant within cells (like ribosomal RNA). Through the use of an enzyme called reverse transcriptase (RT) and primers composed of deoxy-Thymidines ( oligo-dT or dT18), mRNA can be converted into a single strand of DNA that is complimentary to the mRNA. This complimentary DNA is called cDNA . cDNA is very stable compared to the highly labile mRNA and is used for subsequent processing.


Prokaryotic vs Eukaryotic Transcription

Transcription is a process by which the genetic information present in the DNA is copied to an intermediate molecule (RNA). The sequence in the RNA is complementary to that of the gene which is transcribed and thus the RNA retains the same information as the gene itself. Transcription is a universal process in the living word and it occurs both in prokaryotes and eukaryotes. Even though the overall process of transcription is similar in both prokaryotes and eukaryotes, there do exists some fundamental differences between these groups.

This post summarizes the overall similarities and differences between the Prokaryotic and Eukaryotic transcription in a detailed but easy way.

Similarities between prokaryotic and eukaryotic transcription

1. In both groups DNA acts as the template for RNA synthesis
2. In both groups transcription produces RNA molecule
3. Chemical composition of transcript is similar in both groups

4. Transcription is facilitated by the enzyme RNA polymerase in both groups

5. In both groups, one strand of the DNA duplex acts as the template

Difference between prokaryotic and eukaryotic transcription


Watch the video: Eukaryotic Transcription (January 2023).