The synthesis of mRNA from the DNA is called transcription.In this process an RNA copy of a DNA sequence is produced with the help of RNA polymerase.To understand the mechanism behind the transcription process it is useful to focus first on RNA polmerase.
RNA Polymerase
RNA polymerase is best understood in bacteria.RNA polymerase is very large and complex.It consists of six sub_units_two identical alpha (a) and one chain of each of β,β′, omega(ω) and sigma(σ) sub_units.
The complete RNA polymerase enzyme is termed as holoenzyme and can be represented as a2ββ'ωσ and if sigma is removed from holoenzyme then it will become core polymerase.The sigma factor is required for the enzyme to recognize the correct signals on DNA and to initiate synthesis.It is only sigma sub_unit whose attachment is not so firm.Once RNA synthesis is initiated the sigma factor dissociate from the holoenzyme and core enzyme brings about elongation of mRNA (or any other RNA)
Two a(alpha) sub_units bind regulatory protein proteins, a β sub_unit bins RNA nucleoside sub_units.
In both bacteria and eukaryotes, the polymerase adds ribonucleotides to the growing 3′ end of an RNA chain. No primer is needed, and synthesis proceeds in the 5′→3′ direction.In prokaryotes all types of RNA are transcribed by the same enzyme.While eukaryotes have three different polymerases.
Only one of the two strands of DNA, called the template strand, is transcribed. The RNA transcript’s sequence is complementary to the template strand. The strand of DNA that is not transcribed is called the coding strand. It has the same sequence as the RNA transcript, except T takes the place of U. The coding strand is also known as the sense (+) strand, and the template strand as the antisense (–) strand.
The following three stages are commonly distinguished in transcription by RNA poly_merase:(1) binding to promoters and chain initiation, (2) elongation, and (3) termination
RNA Polymerase
RNA polymerase is best understood in bacteria.RNA polymerase is very large and complex.It consists of six sub_units_two identical alpha (a) and one chain of each of β,β′, omega(ω) and sigma(σ) sub_units.
The complete RNA polymerase enzyme is termed as holoenzyme and can be represented as a2ββ'ωσ and if sigma is removed from holoenzyme then it will become core polymerase.The sigma factor is required for the enzyme to recognize the correct signals on DNA and to initiate synthesis.It is only sigma sub_unit whose attachment is not so firm.Once RNA synthesis is initiated the sigma factor dissociate from the holoenzyme and core enzyme brings about elongation of mRNA (or any other RNA)
Two a(alpha) sub_units bind regulatory protein proteins, a β sub_unit bins RNA nucleoside sub_units.
In both bacteria and eukaryotes, the polymerase adds ribonucleotides to the growing 3′ end of an RNA chain. No primer is needed, and synthesis proceeds in the 5′→3′ direction.In prokaryotes all types of RNA are transcribed by the same enzyme.While eukaryotes have three different polymerases.
- RNA polymerase 1 synthesis rRNA and present in the nucleolus
- RNA polymerase II synthesizes mRNA and it is present in nucleoplasm
- RNA polymerase III synthesizes tRNA and also present in nucleoplasm.
Only one of the two strands of DNA, called the template strand, is transcribed. The RNA transcript’s sequence is complementary to the template strand. The strand of DNA that is not transcribed is called the coding strand. It has the same sequence as the RNA transcript, except T takes the place of U. The coding strand is also known as the sense (+) strand, and the template strand as the antisense (–) strand.
The following three stages are commonly distinguished in transcription by RNA poly_merase:(1) binding to promoters and chain initiation, (2) elongation, and (3) termination
Promoter
The first step in transcription is binding of RNA polymerase to a DNA molecule.Binding occurs at particular sites,called the promoters.A promoter is a short sequence that is not itself transcribed by the polymerase that binds to it.Two special promoter regions have been identified that appear in all organisms.In a region of five to ten bases preceding the coding region is a sequence of seven bases that reads:TATAATG with minor variations.A sequence such as this is called a consensus sequence.In bacteria this region is called the Pribnow box and it is located _1o nucleotides upstream of the start site and in the eukaryotes the same region has the sequence TATAAAT and is called the Hogness box and this region is referred as the TATA box.while according to some other books the TATA box is located at _25 and is very similar to the prokaryotic _10 sequence,and , a TTGACA sequence called the –35 sequence, located 35 nucleotides upstream of the position where transcription actually starts and it is present in prokaryotes while in eukaryotes following box are present CAAT box(ACAATCT present in between _70 to _80 nucleotides) and GC box (GGGCGG in between _60 to 100)
It has been suggested that CAAT and GC boxes determine the efficiency of transcription,while TATA box aligns RNA polymerase at proper sites with the help of proteins,called transcription factor or TFs (e.g.,TF II A, TF II B, TF II D).
Transcription factor B&D form a complex known as DB complex
Eukaryotic promoters also consist of sites located 100 to 200 base pair upstream,which interact with proteins other than RNA polymerase,and,thus regulate the activity of promoter.These sites are known as enhancers.
There are other regulatory sites known as silencers which repress gene expression.Both enhancers and silencers can function at great distance from the genes they enhance and repress respectively
Promoters differ widely in efficiency. Strong promoters cause frequent initiations of transcription, as often as every 2 seconds in some bacteria. Weak promoters may transcribe only once every 10 minutes.
 |
| boxes |
It has been suggested that CAAT and GC boxes determine the efficiency of transcription,while TATA box aligns RNA polymerase at proper sites with the help of proteins,called transcription factor or TFs (e.g.,TF II A, TF II B, TF II D).
Transcription factor B&D form a complex known as DB complex
Eukaryotic promoters also consist of sites located 100 to 200 base pair upstream,which interact with proteins other than RNA polymerase,and,thus regulate the activity of promoter.These sites are known as enhancers.
 |
| showing enhancer |
There are other regulatory sites known as silencers which repress gene expression.Both enhancers and silencers can function at great distance from the genes they enhance and repress respectively
Promoters differ widely in efficiency. Strong promoters cause frequent initiations of transcription, as often as every 2 seconds in some bacteria. Weak promoters may transcribe only once every 10 minutes.
Initation
The binding of RNA polymerase to the promoter is the first step in gene transcription. In bacteria, a subunit of RNA polymerase called σ (sigma) recognizes the –10 sequence in the promoter and binds RNA polymerase there. Importantly, this subunit can detect the –10 sequence without unwinding the DNA double helix. In eukaryotes, the –25 sequence plays a similar role in initiating transcription, as it is the binding site for a key protein factor. Other eukaryotic factors then bind one after another, assembling a large and complicated transcription complex.
Transcription factors
The transcription factors whose types are already described,these are proteins which are needed for transcription but are not part of the RNA polymerase.They help in DNA binding of a RNA polymerase to constitute the so_called pre_initiation complex or transcription complex.After the formation of this complex initiation of transcription occurs.All known transcription factors may recognize either DNA sequences,another factor or RNA polymerase.Elongation
There are certain accessory proteins of transcription,called elongation factors,which enhance the overall activity of RNA polymerase II and lead to increase in the elongation rate.At least two such proteins are known (1)The TF II F (2)The TF II S.
 |
| showing transcription bubble |
The transcription of the RNA chain usually starts with ATP or GTP. One of these forms the 5′ end of the chain, which grows in the 5′→3′ direction as ribonucleotides are added. Unlike DNA synthesis, a primer is not required. The region containing the RNA polymerase, DNA, and growing RNA transcript is called the transcription bubble because it contains a locally unwound “bubble” of DNA.
Within the bubble, the first 12 bases of the newly synthesized RNA strand temporarily form a helix with the template DNA strand.The RNA-DNA hybrid helix rotates each time a nucleotide is added so that the 3′ end of the RNA stays at the catalytic site. The transcription bubble moves down the DNA at a constant rate, about 50 nucleotides per second, leaving the growing RNA strand protruding from the bubble. After the transcription bubble passes, the now transcribed DNA is rewound as it leaves the bubble. Unlike DNA polymerase, RNA polymerase has no proofreading capability.
Termination
At the end of a gene are “stop” sequences or signals. The simplest stop signal is a series of GC base-pairs followed by a series of AT base-pairs. The RNA transcript of this stop region forms a GC hairpin followed by four or more U ribonucleotides.The hairpin causes the RNA polymerase to pause immediately after the polymerase has synthesized it, placing the polymerase directly over the run of four uracils. The pairing of U with DNA’s A is the weakest of the four hybrid base-pairs and is not strong enough to hold the hybrid strands together during the long pause. Instead, the RNA strand dissociates from the DNA within the transcription bubble, and transcription stops. A variety of protein factors aid hairpin loops in terminating transcription of particular genes.
Post_transcriptional Modifications
The immediate product of transcription of mRNA in eukaryotes is a molecule of many more ribonucleotides than that comprising the ultimate functional mRNA.This primary transcript may range from 500 to 50,000 nucleotides; it remains confined to the nucleus and is called heterogeneous nuclear RNA (hnRNA).
This hnRNA has long sequences of bases which were transcribed from DNA and there these were named as interons.these interons do not contain meaningful information for protein synthesis.While the area of DNA which had meaningful information and know have transcribed into mRNA is called exerons.
RNa processing
The pre_mRNA undergoes procesing which has following stages
RNA splicing
In RNA splicing the interons are removed and some other modifications are done
In eukaryotes, every mRNA transcript must travel a long journey out from the nucleus into the cytoplasm before it can be translated. Eukaryotic mRNA transcripts are modified in several ways to aid this journey:
Addition of a cap
During capping process, a cap of a methylated guanosine, called 7_methyl_guanosine , is added to 5' end of primary transcript.Capping occurs shortly after initiation of synthesis of the mRNA, possibly before RNA polymerase II leaves the initiation site.II
The biological significance of capping is that, this structure protects the 5′ end of the RNA template from nucleases and phosphatases during its long journey through the cytoplasm. Without these caps, RNA transcripts are rapidly degraded.Caps also help in recognition of ribosomes.
Addition of poly-A tails
The 3′ end of eukaryotic transcript is cleaved off at a specific site, often containing the sequence AAUAAA. A special poly-A polymerase enzyme then adds about 250 A ribonucleotides to the 3′ end of the transcript.This process is called polyadenylation.This long string of As protects the transcript from degradation by nucleases. It also appears to make the transcript a better template for protein synthesis.
Nice job
ReplyDelete