In DNA transcription steps in eukaryotes post we have briefly explained about what is DNA transcription?, enzymes in DNA transcription, signals for initiation of DNA transcription, steps of eukaryotic transcription, initiation of DNA transcription, elongation process of DNA transcription, termination of DNA transcription and post-transcriptional processing.
In 2006, Roger Kornberg was awarded the Nobel Prize for his research on the molecular basis of DNA transcription. He is the son of Arthur Kornberg, who researched on DNA replication and won the Nobel Prize in 1959. After initiation, elongation, and termination, transcription is followed by post-transcriptional processing.
It is one among the first steps in the gene expression process. The passage of genetic information from DNA to protein occurs in a sequential process of transcription and translation. During transcription, just one strand of DNA, known as the template strand, is copied, and the RNA produced is known as mRNA.
Enzymes in DNA Transcription
The RNA polymerase is the main enzyme involved in transcription. It uses single-strand DNA to synthesize a complementary RNA strand.
In higher species, there are three distinct DNA-dependent RNA polymerases (RNAP). Their molecular weight ranges from 500 to 600 kDa. RNAP type II or B, The major enzyme responsible for mRNA synthesis is RNAP type II or B. Alpha amanitin prevents it from working.
RNAP type I or A, Amanitin does not block RNAP type I or A, which is responsible for ribosomal RNA synthesis. RNAP type III or C, RNAP type III or C is in charge of tRNA synthesis and is relatively sensitive to amanitin.
Two alpha, two beta, one omega, one sigma factor, and two zinc molecules make up the bacterial RNAP enzyme. At the start site, the beta subunit fixes. The sigma factor recognises the promoter site and boosts the holo-affinity enzymes for the promoter site.
Steps of Eukaryotic Transcription
Signals for Initiation
There are particular places on the DNA that act as initiators for the initiation process. The RNAP binds to the template DNA strand’s promoter site. On the whole DNA of humans, there are around 105 transcription start sites.
A sequence of 5′-TGTTGACA-3′ can be found about 35 bp upstream of the transcription start site in bacteria. Another nucleotide, 5′-TATAAT-3′, is around 10 bp upstream. The TATA box, also known as the Pribnow box, is the second type. The TATA box is on the coding strand, not the template strand.
In mammals, the TATA box sequence is slightly altered (TATAAA) and is referred to as the Golberg-Hogness box. It is situated between –25 and –30 degrees. It serves as a start region signal. GGCCAATCT, also known as CAAT box, is found further upstream, between –70 and –80. Because they are close to the gene, these recognition signals are said to be cis-acting.
Enhancers and Silencers
Enhancers increase the rate of transcription and silencers decrease the rate. Other regulatory signals for transcription are Hormone response elements (HRE), Repressors, Inducers and Derepressors.
Initiation of Transcription
Steps of eukaryotic transcription: Initiation of Transcription
The starting point of transcription corresponds to the 5′ nucleotide of the DNA, designated as +1. Then numbering is done 2, 3, 4, etc. to the downstream region of the DNA. The nucleotide adjacent to downstream of the starting point is numbered as –1. Further upstream, these negative numbers are increased.
With the help of sigma factor, the DNA helix partially unwinds, and the RNAP attaches to the promoter region on DNA. This is referred to as the pre-initiation complex.
The first nucleotide of the mRNA connects to the initiation site on the beta subunit of RNAP when it reaches the relevant place on the gene. The 5′ end of the mRNA is formed as a result of this. It will be complementary to the nucleotide at that location in the DNA. This is where transcription begins. A purine ribonucleotide is usually the initial unit of nascent mRNA.
The RNAP is then attached to the next nucleotide. There is the formation of a phosphodiester bond. Then the enzyme moves to the next base on the template DNA.
The RNAP undergoes a conformational change and travels away from the promoter area after polymerizing 10-20 nucleotides. This process is known as promoter clearing. The gene has been fully transcribed and Sigma factor has been released.
Steps of eukaryotic transcription: Initiation of Transcription in mammalian system
In eukaryotes, the situation is more complex. There are at least 7 transcription factors, collectively called as Tf-II. First, the TATA box is recognized by TBP (TATA binding protein). Instead of the sigma factor, SL1 factor ensures that RNAP could locate the start point. In humans about 105 transcription initiation sites are available.
Elongation of Transcription
The RNAP is a protein that moves along the DNA template. According to the base pairing rule, new nucleotides are integrated into the nascent mRNA one by one. As a result, A in DNA becomes U in mRNA, T becomes A, G becomes C, and C becomes G. The 5′ to 3′ end of mRNA is where it is synthesised. This signifies that template DNA is read from 3′ to 5′. In DNA synthesis, this is comparable to polarity.
The DNA helix unwinds downstream and winds upstream as the RNAP travels across the DNA template. DNA unwinding is a characteristic of RNAP. This unwinding process will also benefit from topo-isomerase. A transcription bubble is generated, including RNAP, DNA, and nascent RNA. This bubble is around 20 bp long.
There is no proof reading since RNAP has no nuclease activity. As a result, fidelity is worse; the rate of error in mRNA transcription is 104 or 105 times higher than in DNA replication. But it’s not as bad because the errors aren’t passed down to the daughter cells or the following generation.
Steps of eukaryotic transcription: Elongation of Transcription
Termination of Transcription
Steps of eukaryotic transcription: Termination of Transcription
A termination protein called the Rho factor recognises the specific signals. The process of attachment is ATP-dependent. The RNAP is unable to move after it binds to the DNA. As a result, the enzyme separates from DNA, releasing freshly produced mRNA.
There’s also a section on Rho-independent termination. Termination signals are found far downstream of the coding sequence in humans, usually 1000 to 2000 bases distant.
The mRNA formed and released from the DNA template is known as the primary transcript. It is also known as heteronuclear mRNA or hnRNA. In bacteria, mRNA is not changed; and translation of mRNA starts even before completion of transcription.
Post-transcriptional processing is not only for mRNA but for tRNA and rRNA as well. In mammalian system, it undergoes extensive processing to become the mature mRNA.
The 3′ terminus is polyadenylated in the nucleoplasm. This poly-A tail may be 20 to 250 nucleotides long. This tail protects mRNA from attack by 3′ exonuclease.
Capping at 5' End
Eukaryotic mRNAs are all ‘capped’ at the 5′ terminus by 7-methyl guanosine triphosphate. An unusual 5′ to 5′ triphosphate bridge is seen. This is also done inside the nucleus. The cap is useful in recognition of mRNA by the translating machinery.
Methylations of N6 of adenine residue and 2′-hydroxyl group of ribose are common. These are mainly done in the cytoplasm.
The non-coding sequences, i.e., the introns are removed by spliceosome excision. The coding sequences or the exons join together by ligation.