TRANSCRIPTION

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The production of RNA copies from a DNA template is known as transcription. It is catalysed by a specific enzyme RNA polymerase or transcriptase. During this process, only one strand of DNA duplex is known as template strand or antisence strand. This results into the production of m-RNA molecule having base sequence complementary to the template DNA strand. It should be noted here that the sense. strand or coding strand of DNA is now copied and has the same base sequence as the RNA produced by the antisense strand.

The RNA polymerase is a complex enzyme and usually consists of a larger protein part (apoenzyme), which is known as core enzyme and a cofactor, which is known as sigma factor. The two combines to produce the complete enzyme of holoenzyme. Unless and until the two parts of RNA polymerase do not combine with each other, it is not functional. As far as the nature of RNA polymerase in prokaryotes and eukaryotes is concerned, it shows much diversity. While in prokaryotes like E.coli a single species of this enzyme is found, at least three distinct RNA polymerases have been reported in nuclei of most of eukaryotes. These have been named as : 1. RNA polymerase-I or A, 2. RNA polymerase-II or B and 3. RNA Polymerase-III or C. They have different functions as:

RNA polymerase-I or A: It is located in the nucleolus and responsible for the synthesis of rRNA.

RNA polymerase-II or B: It is found in the nucleoplasm and is responsibe for the synthesis of hnRNA which is a precursor of mRNA.

RNA polymerase-III or C: It is also found in the nucleoplasm. It is responsible for the production of 5s rRNA and tRNA.

1.a  Promoters for RNA PolymeraseI. Promoters for RNA polymerase I have atleast two elements:

A GC-rich upstream (-180 to -107) control element

A core region that overlaps the transcription start site (-45 to +20).

Protein coding structural genes in higher eukaryotes are transcribed in the nucleus, but the primary RNA transcripts in the nucleus differ from mRNAs used in the cytoplasm for translation. The RNA transcripts in the nucleus are collectively described as heterogeneous nuclear RNA or pre-mRNA molecules each of which is generally much larger than its corresponding mRNA. The hnRNA molecules, which are destined to produce mRNA, undergo RNA processing which includes the following events: (i) Modification of 5’ end by capping and modification of 3’ end by a tail after enzymatic cleavage; (ii) Splicing out of intron sequences from RNA transcripts of interrupted genes. Cleavage and polyadenylation usually preceed RNA splicing.

Promoter, enhancer and silencer sites for initiation of transcription in eukaryotes

In eukaryotes there are three RNA polymerases: RNA polymerase I or RNAPI for synthesis of pre-rRNA; RNA polymerase II or RNAPII for synthesis of re-mRNA or hnRNA and several snRNAs, and RNA polymerase III or RNAPIII for synthesis of 5S RNA, tRNA. Different promoter sequences have been identified for different RNA polymerases.

MECHANISM OF TRANSCRIPTION: The overall process of transcription is completed in following steps:

1. Formation of holoenzyme: The core enzyme of RNA polymerase cannot start the polymerization process producing RNA. It first combines with the sigma factor and produce the holoenzyme, It is assumed that the sigma factor helps the enzyme in recognition of the initiation site on the DNA template.

2. Attachment of holoenzyme on DNA duplex: The holoenzyme first binds at the promoter site of DNA forming the closed promotor complex or ‘closed binary complex’. In this stage the DNA still remains in the form of double helical.

3. Unwinding of DNA: It includes strand separation in the DNA duplex in a stretch of the DNA bound with RNA polymerase; It extends just beyond the start point so that the template becomes available for transcription initiation. The open DNA strands form the ‘open binary complex’

4. Synthesis of RNA: After the open binary complex is formed on DNA, synthesis of RNA starts. Once the template or antisense strand of DNA becomes available, the enzyme begins to incorporate RNA nucleotides beginning at the start points. The polymerization of these nucleotides takes place in 5’à 3’ direction. As the enzyme molecule move ahead in this direction, phosphodiester linkage or bond is formed between two adjacent nucleotides.

The process of elongation of RNA synthesis take place when the holoenzyme leave the promoter region and move ahead in 5’à3’ direction. Together with the movement of the holoenzyme, the trancription bubble also moves in the same direction. The transcription bubble represents the region of the DNA duplex in which the two strands are separated from each other. The length of the bubble ranges from 12 to 20 base pairs. The bubble movement and sequential adding of correct nucleotides on RNA chain take place simultaneously. The 5’ end of the newly synthesized RNA progressively separate from the DNA template DNA. In the back of the bubble, the two DNA strands reassociate to form DNA duplex.

5. Termination of RNA formation: Specially in prokaryotes, termination of transcription or RNA formation is brought about by certain termination signals on DNA The termination may be of two types:

Rho Independent Terminations: This types of RNA synthesis termination is due to specific sequences on DNA. A typical hairpin like structure is formed on DNA template due to which the movement of RNA polymerase on the template is obstructed. The hairpin structure is formed due to inverted repeat sequences on DNA. The hairpin or stem-loop is followed by a run of adenine residues in DNA and U residues in mRNA in the downstream.

Rho Dependent Termination: This type of termination is due to presence of special factor, which is called Rho factor.  It has a mol. Wt. Of 60,000 and is not a part of RNA polymerase. After the synthesis of mRNA on template DNA is completed, it attaches with the template. The site ofr its attachment is characterized by 5’-CAATCAA-3’. The actual and precise mechanism of the function of factor is not known.

In eukaryotes, the termination process is more completed. The termination sites similar to prokaryotes are also operative in eukaryotes but these sites are believed to be present away up to 1 kb from the site of the 3’end of the mRNA. AAUAAA sequence on mRNA and ‘snurp’ are assumed to play important role in termination of the process in eukaryotes.

Maturation of mRNA from hnRNA in eukaryotes: The mature mRNA molecules very often have much lower molecular wt. And base sequence length in comparison to the DNA segment from which it is transcribed. The primary RNA transcript of a structural gene is called pre-mRNA. It is also known as the heterogeneous RNA, high molecular wt. RNA. It is much bigger in size than mRNA.

The later is formed by splicing of hnRNA followed by some other modifications.

The heteronuclear mRNA undergoes following modifications: -

Addition of Cap (m⁷G) and Tail (Poly A) for mRNA in Eukaryotes

1. Addition of methylated cap at the 5’ end

The initial RNA transcript, derived from genes coding for proteins, gets modified so that its 5’ end gains a methylated guanine and its 3’ end is polyadenylated. Capping at 5’ end occurs rapidly after the start of transcription and much before completion of transcription. Transcription starts with a nucleoside triphosphate, and a 5’ triphosphate group is retained at this first position. The initial sequence at 5’ end of hnRNA is therefore 5’ pppApNpNp…3’. To the 5’ end is added a terminal G with the help of an enzyme guanyl transferase as follow:

5’Gppp+5’pppApNpNp­ 5’Gppp5’ApNpNp+pp+p

The new G residue is in the reverse orientation with respect to all other nucleotides and undergoes methylation at its 7^th position. The cap with a single methyl group at this terminal guanine residue is found in unicellular eukaryotes and described as cap0, but in most eukaryotes, methyl group may also be present on the penultimate base at 2’ position of sugar moiety, so that nucleotides, it is now described as cap1. Removal of cap leads to loss of translation activity due to loss of the formation of mRNA-ribosome complex. It suggests that the ‘cap’ helps in recognition of ribosome. Only in some eukaryotic nRNAs, caps may be absent and may not be required for translation.

     2.    Polyadenylation and the generation of 3’ end in eukaryotes

The 3’end of n RNA is generated in two steps (i) Nuclease activity cuts the transcript at an appropriate location. (ii) Poly (A) is added to the newly generated end by an enzyme, poly (A) polymerase (PAP), utilizing ATP as a substrate. Ordinarily 30% of hnRNA and 70% of mRNA are polyadenylated. In addition to AAUAAA, there are following consensus sequences, that are involved in polyadenylation: (i) a G-U rich element is present downstream to the site of cleavage, and is important for efficient processing for polyadenylation: (ii)  a G-A sequence immediately5’ of the cleavage site;(iii) consensus upstream element situated 5’ of a poly A signal or AAUAAA.

3. Splicing of RNA parts coded by introns: Self splicing is a very common phenomenon found in hnRNA. In this process generally those parts of the RNA are removed or spliced out, which have been transcribed from intron regions of the template DNA. These regions have short consensus sequences which pair to formstem-loop like secondary structure. These are helpful in self or autosplicing. Stem-loop like structures were observed for the first time in the hnRNA of Tetrahymena thermophila.

4. Editing of RNA: Theoretically, the base sequence of a mRNA is just complementary to the base sequence of the segment of the template DNA from which it is transcribed. However, in many cases, the base sequence of mRNA has been found to be changed after transcription at the level of RNA. This process of change in the base sequence of mRNA is known as RNA editing. It may be confined to a single base or may affect the entire mRNA.