Steps under Which the Entire Transcription Process may be divided are:
1. Pre-initiation:
In eukaryotes, RNA polymerase and therefore the initiation of transcription requires the presence of a core promoter sequence in the DNA.
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Promoters are regions of DNA which promote transcription and in eukaryotes are found at -30, -75 and – 90 base pairs upstream from the start site of transcription.
Core promoters are sequences within the promoter which are essential for transcription initiation.
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RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors.
The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box found -30 base pairs from the start site of transcription.
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The TATA box as a core promoter is the binding site for a transcription factor known as TATA binding protein (TBP), which is itself a subunit of another transcription factor called Transcription Factor II D (TFIID).
After TFIID binds to the TATA box via the TBP, five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a pre-initiation complex.
One transcription factor, DNA helicase, has helicase activity and so is involved in the separating of opposing strands of double-stranded DNA to provide access to a single-stranded DNA template.
However, only a low or basal, rate of transcription is driven by the pre-initiation complex alone. Other proteins known as activators and repressors, along with any associated coactivators or co repressors, are responsible for modulating transcription rate.
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Thus pre- initiation complex contains: 1.Core Promoter Sequence 2.Transcription Factors 3.DNA Helicase 4.RNA Polymerase 5.Activators and Repressors.
The transcription pre-initiation in archaea is essentially homologous to that of eukaryotes, but is much less complex.
The archaeal pre-initiation complex assembles at a TATA-box binding site however, in archaea this complex is composed of only RNA polymerase II, TBP, and TFB (the archaeal homologue of eukaryotic transcription factor II B (TFIIB).
2. Initiation:
In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. RNA polymerase is a core enzyme consisting of five subunits: 2 a subunits, 1 B subunit 1 13′ subunit, and 1 u subunit.
At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 base pairs downstream of promoter sequences.
Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase does not directly recognize the core promoter sequences.
Instead, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription.
Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it.
The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex. Transcription in the archaea domain is similar to transcription in eukaryotes.
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. Abortive initiation continues to occur until the o factor rearranges resulting in the transcription elongation complex (which gives a 35 bp moving footprint).
The o factor is released before 80 nucleotides of mRNA are synthesized. Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur.
This, like most of the remainder of transcription is an energy-dependent process, consuming adenosine triphosphate (ATP).
Promoter clearance coincides with phosphorylation of serine 5 on the carboxy terminal domain of RNA Pol in eukaryotes which is phosphorylated by TFIIH.
3. Elongation:
One strand of the DNA, the template strand (or non-coding strand), is used as a template for RNA synthesis.
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 polymerase transverses the template strand from 3′ – 5′, the coding (non-template) strand and newly- formed RNA can also be used as reference points, so transcription can be described as occurring 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).
Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA) so many mRNA molecules can be rapidly produced from a single copy of a gene.
Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases.
In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure.
4. Termination:
Bacteria use two different strategies for transcription termination. In Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop followed by a run of Us.
When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA-RNA hybrid.
This pulls the poly-U transcript out of the active site of the RNA polymerase, effectively terminating transcription.
In the “Rho-dependent” type of termination, a protein factor called “Rho” destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.
Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template- independent addition of As at its new 3′ end in a process called polyadenylation.
Proteins are linear polymers of individual building blocks called amino acids. The sequence of bases along the DNA strand determines the sequence of the amino acids in proteins.
There are 20 different amino acids in proteins but only 4 different bases in DNA (A, T, C and G). Each amino acid is specified by a codon, a group of three bases. Because there are 4 bases in DNA, a three- letter code gives 64 (4 x 4 x 4) possible codons.
These 64 codons form the genetic code the set of instructions that tells a cell the order in which amino acids are to be joined to form a protein.
Despite the fact that the sequence of codons on DNA determines the sequence of amino acids in proteins, the DNA helix does not itself play a role in protein synthesis.
The translation of the sequence from codons into amino acids occurs through the intervention of members of a third class of molecule messenger RNAs (mRNA).
Messenger RNA acts as a template guiding the assembly of amino acids into a polypeptide chain.
Messenger RNA uses the same code as the one used in DNA with one difference: In mRNA the base uracil (U) is used in place of thymine (T).
When we write the genetic code, we usually use the RNA format i.e. we use U instead of T.