In this article we will discuss about:- 1. Introduction to Transcription in Eukaryotes 2. Mechanism of Transcription in Eukaryotes 3. Transcription Factories 4. Reverse Transcription 5. Role.
Introduction to Transcription in Eukaryotes:
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.
2. Template used:
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.
3. Enzyme Involved:
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.
5. First Step:
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:
(3) Promoter clearance,
(4) Elongation and
These are briefly discussed as follows:
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 ~8 polymerases.
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.