gene expresion 2

8/9/2019 Gene Expresion 2

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GENE EXPRESSION

VIKRANT JOSHI

F.Y. BIOTECHNOLOGY

6519

KHALSA COLLEGE

AMRITSAR


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The biological information of the cells is stored in its DNAmolecule. Gene expression is the process by which this

information stored in DNA is made available to the cell tosynthesize protein.

Crick proposed that the use of this information is describedby central dogma which states that information istransferred from DNA to RNA to protein.

During gene expression, DNA molecules copy theirinformation by directing the synthesis of an RNA moleculeof complementary sequence. This process is k/atranscription.

DNA RNA PROTEIN

transcription translation

CENTRAL DOGMA


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The RNA then directs the synthesis of a polypeptide whoseamino acid sequence is determined by the base sequence ofthe RNA. This process is k/a translation.

Central dogma states that the transfer of information canonly occur in one direction i.e from DNA to RNA to protein.

An exception to this is found in retrovirus which have anenzyme called reverse transcriptase which can copy RNAinto DNA.


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Deoxyribonucleic acid, or

DNA, is a nucleic acid thatcontains the geneticinstructions used in thedevelopment & functioningof all known livingorganisms.

DNA is a polymer consistsof long chain of monomers,k/a nucleotides.

Nucleotide has 3 parts:

1. A sugar (2 deoxyribose)

2. Nitrogen containing ring

structure: base (Adeninie,guanine, thymine & cytosine)

3. A phosphate group


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Sugar plus a base is k/anucleoside (bond is between1 C of sugar & N at 9th

position in purine & 1

st

inpyrimidine). When phosphate group gets

attached to nucleoside, it isk/a nucleotide. PO4 isattached to 5 C of sugar.

5 phosphate of one

nucleotide forms aphosphodiester bond withthe 3 carbon of the nextnucleotide eliminating the OH group. Thus apolynucleotide chain isformed.

This polynucleotide has afree 5 triphosphate at oneend k/a 5 end and a free 3OH group at the other endcalled the 3 end. So DNAmolecule runs as 35 or 53.


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DNA molecules have a very distinct 3-D structure k/adouble helix. This was discovered by Waston & Crick.

DNA exists as 2 polynucleotide chains wrapped around eachother to form the double helix.

The sugar-phosphate part of the molecule forms a spine orbackbone.

The double helix execute a turn after every 10 base pairs. The double helix is said to be anti-parallel. One of the

strand runs in 35 direction & the other in 5 3direction.

Major grooves & minor grooves for interaction withproteins. The double helix is right handed.

Different types of DNA has been identified. B from: in cells.

A, C, D, E & Z are left handed. Complementary base pairing: The DNA double helix is

stabilized by hydrogen bonds between the bases attachedto the two strands.


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Ribonucleic acid or RNA is a nucleic acid polymerconsisting of nucleotide monomers that plays severalimportant roles in the processes that translate geneticinformation from (DNA) into protein products.

RNA is very similar to DNA, but differs in a few importantstructural details:

1. RNA nucleotides contain ribose sugars while DNAcontains deoxyribose.

2. RNA uses predominantly uracil instead of thyminepresent in DNA.

There are 3 major types of RNA that participate in theprocess of protein synthesis.

Ribosomal (rRNA), transfer (tRNA) & messenger(mRNA).


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Ribosomal RNA is found in association with a number ofdifferent proteins as components of ribosomes.

There are three distinct types of rRNA (23s, 16s &5s) inprokaryotic cells & eukaryotic mitochondria .

In eukaryotic cytosol, there are four types of rRNA(28s,18s,5.8s,5s).

The function of the rRNA is to provide a mechanism fordecoding mRNA into amino acids and to interact with thetRNAs during translation.


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Messenger RNA consists ofabout 5% of RNA & itcarries the geneticinformation from DNA tocytosol where it is used as

the template for proteinsynthesis.

The "life cycle" of an mRNAin a eukaryotic cell is RNAis transcribed in thenucleus, once completely

processed, it is transportedto the cytoplasm andtranslated by theribosome.


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Transfer RNA, thesmallest of three majortypes has a cloverleaf

structure which consistsof a series of stem loopstructures known asarms.

These include:1. acceptor arm2. D or DHU arm

(dihydrouracil)3. anticodon arm4. extra or optional arm5. TC arm (pseudouracil). Function of tRNA is to link

the nucleotide sequences

of mRNA to the aminoacid sequences of polypeptides.

C

C

A

Aminoacid

bindingsite

Anticodon arm:recognizing &binding codonsin mRNA


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Transcription: is the process through which a DNAsequence is enzymatically copied by an RNA polymerase

to produce a complementary RNA. It is the transfer ofgenetic information from DNA into RNA.

It occurs in 3 steps:

1. Initiation

2. Elongation

3. Termination. Template: a single strand of DNA acts as a template or

coding strand to direct the formation of complementaryRNA during transcription. The strand that is not used asthe template is called the non-coding/sense/+ strand.

Substrate: 4 ribonucleoside triphosphate i.e ATP, GTP,CTP & UTP.

Direction of synthesis: subsequent nucleotides are addedto 3 OH gp of preceding nucleotide. Therefore, RNAchain growth proceeds in 3 to 5 direction.


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Enzymes:

1. prokaryotes have a single RNA polymerase responsible forall cellular RNA synthesis. The structure of this enzyme is:a core enzyme (consisting of 2 , & subunits) &

holoenzyme (core enzyme plus subunit).2. Eukaryotes have one mitochondrial & 3 nuclear RNA

polymerases: I, II & III.


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INITIATION:

Promoter sequence:

Transcription can not start randomly but must begin atspecific sites k/a promoter sequence. It contains specificDNA sequences that act as point of attachment for theRNA polymerase.

Prokaryotic promoters:

2 sequence elements are recognized: -10 sequence (also

k/a pribnow box: TATAAT) & -35 sequence (TTGACA). Eukaryotic promoters:

The promoters used by RNA polymerase I & III aresimilar to prokaryotic promoters.

For RNA polymerase II:

1. TATA box (Hogness box) having sequence TATA.2. CAAT (CCAAT)

3. GC (GGGCG)


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Initiation factors: for initiation of transcription.

In prokaryotes:

The subunit of RNA polymearse is responsible forrecognizing & binding to the promoter sequence, probablyat -35 box.

When enzyme binds with the promoter, it forms closedpromoter complex within which DNA is in the form ofdouble helix. This enzyme covers about 60 base pairsincluding -10 & -35 boxes.

To allow transcription to begin, double helix partiallydissociates at -10 box, to give an open promoter complex.

The subunit then dissociate from open promoter complexleaving the core enzyme.

At the same time the enzyme catalyzes the formation ofphosphodiester bond between first 2 bases & transcriptionis initiated.


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Rifampin is an effective antibacterial drug & one of the

few therapeutic drugs that affect only transcription. Itsaction is:

It binds with to subunit of RNA polymerase & inhibitsthe initiation of transcription. It has no effect oneukaryotic nuclear RNA polymerases.

In eukaryotes: Multiple factors & RNA polymerase II are required to initiate

transcription.

1. Transcription factor IID: contains TATA box-bindingprotein (TBB) that recognizes & binds to the TATA boxsequence independently of RNA polymerase II.

2. Transcription factors TFIIA, TFIIB, TFIIF, TFIIE & TFIIH.


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RNA polymerase II gets attached to the promotersequence.

Attachment is achieved with the help of a series oftranscription factors. These factors are k/a enhancers.

Similar sequences k/a silencers also occur which inhibittranscription.

RNA polymerase is inhibited by -amanitin, a potent toxinproduced by the poisonous mushroom (amanitaphalloides) also k/a death cap or destroying angel. -amanitin forms a tight complex with the polymerase &inhibiting the RNA synthesis & ultimately protein

synthesis.


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Termination:

Prokaryotes & eukaryotes use an identical mechanism of

synthesizing RNA & share many similarities. But very littlecommon in the way they terminate transcription.

The transcription unit extends from promoter to terminatorregion.

In prokaryotes: 2 classes of termination events:

1. Factor independent termination: Termination occurs nonrandomly at specific pointsequences k/a pallindromes. These sequences aresymmetrical about their middle such that the 1st half of thesequence is followed by its exact complement in the 2nd

half.

In single stranded RNA molecule, this feature allows thefirst half of the sequence to base pair with the 2nd half toform a stem loop structure.


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Factor dependent termination:

Particular sequence acts as termination sequence in thepresence of factor rho.

In eukaryotes:

1. RNA polymerase I terminates transcription in a factordependent manner.

2. RNA polymerase III terminates transcription by anunknown mechanism after the synthesis of a series of U-residues.

3. There is no known transcription termination signal for

RNA polymerase II .


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Reverse transcription: Some viruses (such as HI, the cause of AIDS), have the

ability to transcribe RNA into DNA in order to see a cell's

genome. The main enzyme responsible for this type of transcription

is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for

synthesising a complementary DNA strand (cDNA) to theviral RNA genome.

An associated enzyme, ribonuclease H, digests the RNAstrand and reverse transcriptase synthesises acomplementary strand of DNA to form a double helix DNAstructure.

This cDNA is integrated into the host cell's genome viaanother enzyme (integrase) causing the host cell togenerate viral proteins which reassemble into new viralparticles.

Subsequently, the host cell undergoes programmed celldeath (apoptosis).


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Translation is the second process of protein biosynthesis(part of the overall process of gene expression).

Translation occurs in the cytoplasm where the ribosomesare located.

Ribosomes are made of a small and large subunit whichsurrounds the mRNA.

In translation, messenger RNA (mRNA) is decoded toproduce a specific polypeptide according to the rulesspecified by the genetic code.

This is the process that converts an mRNA sequence into achain of amino acids that form a protein.

Translation is necessarily preceded by transcription.

Translation proceeds in four phases: activation, initiation,elongation and termination.


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Translation: process by which cell synthesizes proteins.

During translation, information encoded in mRNA

molecules is used to specify the amino acid sequence of aproteon.

Components required for translation:

Amino acids: all amino acids that are eventually appearin the finished protein must be present at the time of

protein synthesis. If one amino acid is missing, e.g, if thediet does not contain an essential amino acid, that aminoacid is therefore in the limited supply in the cell &translation stops at the codon specifying that amino acid.

Transfer RNA: at least one specific type of tRNA isrequired per amino acid. In human, there are about 50

species of t RNA, whereas bacteria contain 30-40.Because there are only 20 amino acids, some of themhave more than one specific tRNA molecule.


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Amino acid attachment site:3 end is the amino acidattachment site. When tRNAhas covalently attached amino

acid, it is k/a charged & amino acid is k/a activated.

Anticodon: tRNA has 3 basenucleotide sequence k/aanticodon that recognizesspecific codon on mRNA.


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Messenger RNA: specific RNA ia required as template forthe synthesis of the desired polypeptide chain is required.

Amino-acyl-tRNA synthetases: this family of enzymes isrequired for the attachment of amino acids to theircorresponding tRNAs.

Functionally competent ribosomes: ribosomes are large complexes of protein & rRNA.

They consists of 2 subunits: one large & one small.

These are the factories of protein synthesis.

Protein factors: initiation, elongation & termination factors

are required for peptide synthesis. Some of these proteinfactors perform a catalytic function, whereas others appearto stabilizes the synthetic machinery.


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ATP & GTP are required as sources of energy:

Cleavage of 4 high energy bonds are required for the

addition of one amino acid to the growing polypeptidechain.

2 from ATP in amino-acyl tRNA synthetase reaction & 2from GTP.

Codon recognition by tRNA: recognition of a particular

codon in mRNA sequence is accomplished by the anticodonsequence of tRNA.

Anti-parallel binding between codon & anticodon: followsthe rule of complementary & antiparellel binding i.e themRNA codon is read from 5 to 3 by anticodon having 3 to5 orientation.

Wobble hypothesis: describes the mechanism by whichtRNA can recognize more than one codon for a specificamino acid.


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Steps in protein synthesis:

The pathway of protein synthesis is k/a translationbecause the language of the nucleotide sequence onmRNA is translated into the language of an amino acidsequence.

The mRNA is translated from its 5 end to its 3 end,producing a protein synthesized from its amino-terminal

end to its carboxyl-terminal end. Prokaryotic mRNA often have several coding regions that

is they are polycistronic.

Process of protein synthesis is divided into 3 steps:

1. Initiation

2. Elongation3. Termination


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Initiation:

Involves the assembly of the components of the

translation system before peptide bond formation occur. These components involve 2 ribosomal RNA subunits,

mRNA, amino-acyl tRNA , GTP & initiation factors thatfacilitate assembly of this initiation complex.

In prokaryotes, about 3 initiation factors are known (IF-1,IF-2 & IF-3). In eukaryotes, 9 initiation factors arepresent (1-9 eIF).

There are 2 mechanisms by which ribosomes recognizesthe nucleotide sequence that initiate translation.

Shine-Dalgarno sequence:

In E Coli, a sequence of nucleotide bases (5-UAGGAGG)k/a Shine-Dalgarno sequence is located 6-10 basesupstream of the AUG codon on mRNA molecule i.e near 5end.


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The 16S ribosomal RNA component of 30S ibosomal subunithas a nucleotide sequence near its 3 end that iscomplementary to all or part of Shine-Dalgarno sequence.

Therefore, tha mRNA 5 end & 3 end of 16S rRNA can formcomplementary base pairing, thus facilitating tha binding &positioning of the mRNA on the 30S rRNA subunit.

Initiation codon:

The codon AUG at the beginning of the message isrecognized by a special initiator tRNA


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Elongation:

Elongation of the polypeptide chain involves the addition of

amino acids to the carboxl end of the growing polypeptidechain.

As soon as the initiation complex is formed, a largeribosomal subunit binds to it.

The complete ribosome contains 2 binding sites for tRNAmolecules.

The first site is the P/Peptidyl site & is occupied by tRNAmet

base-paired to AUG.

The second site is A/Amino-acyl site & is the positionedover the second codon.

Elongation begins when tRNA enters the A site & base paors

with second codon.

Reaction is catalyzed by peptidyl transferase.


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Termination:

It occurs when one of the 3 termination codons (UAG, UGA,UAA) enters the A site.

Proteins k/a releasing factors enters the A site 7 cause thecompleted polypeptide to be released.

In E Coli, releasing factors are RF1 (UAA & UAG) & RF2(UAA & UGA).

In eukaryotes, a single protein, eRF is involved which

requires GTP for ribosome binding. This is subsequentlyhydrolysed & eRF is released from mRNA & dissociates.

POLYSOMES:

Translation begins at 5 end of mRNA with the ribosomeproceeding along the RNA molecule. Because of the length

of most mRNAs, more than one ribosome at a time cangenerally translate the message. Such a complex of onaemRNA & a number of ribosomes is k/a polysome orpolyribosome.


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Post translational modifications: following translation,

newlysynthesized polypeptides may undergo a range ofmodifications before becoming functional proteins.

These involve mainly the covalent attachment of chemicalgroups & cleavage of the polypeptide chain.

Many different chemical modifications of the side chains of

amino acids or the amino or carboxyl termini of proteinsare found.

Modification may involve the addition of small groups suchas methylation, phosphorylation, acetylation &hydroxylation as well as addition of larger molecules suchas lipids, oligosaccharides.


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gene expresion 2

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