gene expression
TRANSCRIPT
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GENE EXPRESSION
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INTRODUCTIONGENE EXPRESSION It is the process by which a gene's DNA
sequence is converted into the structures and functions of a cell.
Non-protein coding genes are not translated into protein.
Genetic information, chemically determined by DNA structure is transferred to daughter cells by DNA replication and expressed by Transcription followed by Translation.
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• This series of events is called “Central Dogma” is found in all cells and proceeds in similar ways except in retroviruses which posses an enzyme reverse transcriptase which converts RNA into complementary DNA.
• Biological information flows from DNA to RNA , and from there to proteins.
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THE CENTRAL DOGMA OF LIFE
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• Gene expression is a multi-step process which involves
o Replicationo Transcriptiono Translation
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REPLICATION OF DNA
• It is a process in which DNA copies itself to produce identical daughter molecules of DNA.
• DNA strands are antiparallel and complementary, each strand can serve as a template for the reproduction of the opposite strand.
• This process is called semiconservative replication.
• As the newly synthesized DNA has one half of the parental DNA and one half of new DNA.
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• STEPS INVOLVED IN REPLICATION..1. INITIATION.2. ELONGATION.3. TERMINATION
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INITIATION DNA replication starts at specific sites called Origin. A specific dna A protein binds with this site of
origin and separates the double stranded DNA. Separation of two strands of DNA results in the
formation of replication bubble with a Replication Fork on either strands.
A Primer recognises specific sequences of DNA in the replication bubble and binds to it.
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Helicase: The helicase unwinds the DNA helix by breaking the Hydrogen bonds between the base pairs.
Topoisomerase: The topoisomerases introduce negative supercoils and relieve strains in the double helix at either end of the bubble.
The SSB proteins: The SSB proteins (Single Strands Binding) stabilize the single strands thus preventing them to zip back together.
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ELONGATION• DNA polymerase III binds to the Template strand at the 3’
end of the RNA Primer and starts polymerizing the nucleotides.
• On leading strand polymerization of nucleotides proceeds in 5’ – 3’ direction towards the replication fork without interruption.
• Lagging strand is replicated in 5’ – 3’ direction away from replication fork in pieces known as Okazaki Fragments.
• As DNA polymerase reaches the 5' end of the RNA primer of the next Okazaki fragment; it dissociates and re-associates at the 3' end of the primer.
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• DNA polymerase I remove the RNA primers, and fills in with DNA.
• DNA ligase seals the nicks and connects the Okazaki fragments.
• Helicase continues to unwind the DNA into two single strands ahead of the fork while topoisomerases relieves the supercoiling caused by this.
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TERMINATION• Termination occurs when DNA replication forks
meet one another or run to the end of a linear DNA molecule.
• Also, termination may occur when a replication fork is stopped by a replication terminator protein.
• DNA Ligase fills up the gaps between the Okazaki fragments.
• If mistake or damage occurs, enzymes such as a nuclease will remove the incorrect DNA. DNA polymerase will then fill in the gap.
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TRANSCRIPTION
• Transcription is the process through which a DNA sequence is enzymatically copied by an RNA polymerase to produce a complementary RNA or in other words, the transfer of genetic information from DNA into RNA.
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Transcription is divided into 3 stages.• Initiation • Elongation• TerminationINITIATION• RNA polymerase (RNAP) recognises and
binds to a specific region in the DNA called promoter
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• There are two different base sequences on the coding strand which the RNA polymerase recognises and for initiation:
• Pribnow box (TATA box) consisting of 6 nucleotide bases (TATAAT) and is located on the left side about 10 bases upstream from the starting point of the transcription.
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• The ‘-35’ sequence second recognition site in the promoter region of the DNA and contains a base sequence TTGACA which is located about 35 bases upstream of the transcription starting point.
• Closed complex RNAP binds to double stranded DNA and this structure is called Closed complex.
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Open complex After binding of RNAP, the DNA double helix is partially unwound and becomes single-stranded in the vicinity of the initiation site. This structure is called the open complex.
Elongation RNA synthesis then proceeds with addition of
ribonucleotide ATP, GTP, CTP and UTP as building units.
One DNA strand called the template strand serves as the matrix for the RNA synthesis
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• RNAP enzymes transcribe RNA in antiparallel direction 5’ → 3’. Transcription proceeds in complementary way :-
Guanine in DNA leads to Cytosine in RNA Cytosine in DNA leads to Guanine in RNA Thymidine in DNA leads to Adenine in RNA But Thymidine in DNA is replaced by Uracil
in RNA as consequence the Adenine in DNA shows up for Uracil in
RNA.
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• Different types of RNAPs RNA Polymerase I is located in the nucleolus and
transcribes ribosomal RNA (rRNA). RNA Polymerase II is localized to the nucleus,
and transcribes messenger RNA (mRNA) and most small nuclear RNAs (snRNAs).
RNA Polymerase III is localized to the nucleus (and possibly the nucleolar- nucleoplasm interface), and transcribes tRNA and other small RNAs
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• Termination
• Two termination mechanisms are well known :- Intrinsic termination (Rho-independent termination) Terminator sequences within the RNA that signal the
RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template. Example 'GCCGCCG'
The RNA polymerase fails to proceed beyond this point and the nascent DNA-RNA hybrid dissociates.
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Rho-dependent termination uses a termination factor called ρ factor (rho factor) to stop RNA synthesis at specific sites.
This protein binds and runs along the mRNA towards the RNAP. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA and terminates transcription.
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• Post transcriptional modification
• Post transcriptional modification is a process in which precursor messenger RNA is converted into mature messenger RNA (mRNA).
• The three main modifications are
I. 5' capping
II. 3' polyadenylation
III. RNA splicing
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5' capping Addition of the 7 - Methylguanosine cap to 5’ end is the first step in post-mRNA processing. This step occurs co-transcriptionally after the growing RNA strand has reached 30 nucleotides.
3' polyadenylation The second step is the cleavage of the 3' end of the primary transcript following by addition of a polyadenosine (poly-A) tail.
RNA splicing RNA splicing is the process by which introns are removed from the mRNA and the remaining exons connected to form a single continuous molecule. The splicing reaction is catalyzed by a large protein complex called the spliceosome.
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TRANSLATION
It is a process by which proteins are synthesized. Translation is a complex cellular process where mRNA molecules, ribosomes, tRNA molecules, amino acids, aminoacyl synthetases, energy sources ATP and GTP and a number of factors act together in a highly coordinated way.
The mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to the ribosome.
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The ribonucleotides are "read" by translational machinery in a sequence of nucleotide triplets called codons. Each of these triplet codes for a specific amino acid. The ribosome and tRNA molecules translate this code to produce proteins.
tRNAs have a site for amino acid attachment, and a site called an anticodon. These anticodon is an RNA triplet complementary to the codons of mRNA.
Aminoacyl tRNA synthetase catalyzes the bonding between specific tRNAs and the amino acids that their anticodons sequences call for. The product of this reaction is an aminoacyl-tRNA molecule.
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• Initiation Initiation of translation is divided into four
stages:-• Dissociation of Ribosome Initiation starts with the dissociation of the 80s
ribosome into 40s and 60s subunits. Initiation factor IF-3 and IF-1A binds to the 40s
subunit and prevents its re-associaton with 60s subunit.
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• Formation of 43s preinitiation complex The first aminoacyl tRNA (fmet-tRNA) binds
to the 40s ribosomal subunit and forms preinitiation complex. Initiation factor IF3 and IF-1A stabilises this complex.
• Formation of 48s initiation complex mRNA joins to the 43s preinitiation complex
and forms the 48s initaition complex. This step requires energy from ATP.
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Ribosomal initiation complex scans the mRNA for the identification of the appropriate initiation codon and its identification is facilitated by specific sequence of nucleotide surrounding it called Kozak Consensus sequences.
In case of prokaryotes the recognition sequence of initiation codon is referred to as Shine-Dalgarno sequence.
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• Formation of 80s initiation complex Initiation ends as the large 60s ribosomal
subunit joins the 48s initiation complex causing the dissociation of initiation factors.
The binding involves the hydrolysis of GTP. The step is facilitated by the involvement of
IF-5.
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• Elongation
• Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. During elongation the ribosome moves from the 5’ – end to the 3’ – end of the mRNA that is being translated.
• Elongation is divided into Three steps:-
• Binding of aminoacyl-tRNA to A site The 80s initiation complex contains met-tRNA on the
P-site and the A-site is free. Another aminoacyl-tRNA recognises the codon on the
A-site and binds to it. This binding is facilitated by elongation factor-1α and
requires energy from GTP.
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• Formation of peptide bond Now the P site contains the beginning of the
peptide chain of the protein to be encoded and the A site has the next aminoacid to be added.
The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site.
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• Translocation Now, the A site has newly formed peptide,
while the P site has an unloaded tRNA (tRNA with no amino acids).
Then the ribosome moves 3 nucleotides towards the 3' - end of mRNA.
Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A site to the P site and moving the uncharged tRNA to the E exit site. This process is catalyzed by elongation factor EF-2
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• Termination Termination occurs when one of the three
termination codons moves into the A site. These codons are recognized by proteins
called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons).
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• These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome. At the same time the ribosome is dissociate from the mRNA and recycled and used to synthesise another protein.
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• Protein folding Protein folding is the process by which a
protein assumes its characteristic functional shape or tertiary structure, also known as the native state.
All protein molecules are linear heteropolymers composed of amino acids; this sequence is known as the primary structure.
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Most proteins can carry out their biological functions only when folding has been completed, because three-dimensional shape of the proteins in the native state is critical to their function.
The process of folding often begins co-translationally , so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome.
Specialized proteins called chaperones aid in the folding of other proteins.
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• Posttranslational modification
• Many proteins synthesized by translation are not functional as such. Many changes takes place in the protein after synthesis which converts it into active protein. These are known as post transcriptional modifications.
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• Trimming by Proteolytic Degradation Many proteins are synthesized as precursors
which are bigger in size than functional proteins. Some portions of precursors is removed by proteolysis to liberate active protein . This process is called trimming.
Example formation of insulin from proinsulin.
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• Intein splicing Inteins are intervening sequences in proteins.
These are comparable to introns in mRNA. Inteins have to be removed and exteins ligated in the appropriate order for the protein to become active.
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• Covalent Modifications Proteins synthesized by translation are
subjected to many covalent changes. By these changes the proteins are converted to active or inactive form. The covalent changes include many modifications such as Phosphorylation, hydroxylation, Glycosylation, Methylation, Acetylation etc.
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References
1.Biotechnology, by U. Sathyanarayana (page number 38 – 58).
2.The molecular biology of cell by Albert, Johnson,Lewis. 5th edition
3.Net source.
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