GENE EXPR SRS

8/7/2019 GENE EXPR SRS

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VISHAL BABUSHETTY

I- M. Pharm.

Dept. of Pharmacology.

AACP.

Bangalore.


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G

enes are made up of DNA. Each chromosomecontains many genes A gene is the basicphysical and functional unit of heredity. Inhumans, genes vary in size from a few hundred

DNA bases to more than 2 million bases.

What is a gene?


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Every person has two copies of each gene, oneinherited from each parent.

Most genes are the same in all people, but asmall number of genes (less than 1 percent of

the total) are slightly different between people. Alleles are forms of the same gene with small

differences in their sequence of DNA bases.These small differences contribute to each

persons unique physical features


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GeneGene

ExpressionExpression

Gene expression, orexpression, is the

process by which agene's DNA sequence isconverted into thestructures and functions

of a cell. Non-proteincoding genes (e.g. rRNAgenes, tRNA genes) arenot translated into

protein.


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Genetic information, chemically determined by DNA structure is transferred to

daughter cells by DNA replication and expressed by Tancription followed by

Translation. 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.

DNA linked processes can be depicted as

DNAm RNAp Protein

It was found that many genes are expressed differently from what was predicted

by central dogma. Foe example RNA derived from transcription of eukaryotic DNA undergoes splicing.

Proteins are modified after their synthesis to form the active protein.

Thus gene expression is a multi-step process that involves

Replication of DNA.

Transcription.

Post transcriptional modification.

Translation into a gene product .

Folding .

Post-translational Modification.


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Patterns of Gene Expression

Numerous terms are used to describe patterns of gene expression, including:

A constitutive gene is a gene that is transcribed continually compared to a

facultative gene which is only transcribed when needed.

A housekeeping gene is typically a constitutive gene that is transcribed at

a relatively constant level.

A facultative gene is a gene which is only transcribed when needed compared to

a constitutive gene.

An inducible gene is a gene whose expression is either responsive to

environmental change or dependent on the position of the cell cycle.


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Replication

During cell division the Genetic information in a parental cell is transferred to the

daughter cells by DNA replication.

Duplication of an old DNA molecule into two new DNA molecules is called

Replication.

Since 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 DNAhas one half of the parental DNA and one half of new DNA.


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Steps OfReplication

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 replicationbubbleand binds to it.

Helicase: The helicase unwinds and unzips 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 reassociates at the 3' end of the

primer.

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.

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 intoRNA.


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Transcription is divided into 3 stages: initiation, elongation and termination.

Initiation

RNA polymerase (RNAP) recognises and binds to a specific region in

the DNA called promoter.

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.

The -35 sequence second recognition site in the promote r 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.

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.


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Elongation

` RNA synthesis then proceeds with addition of ribonucleotides ATP,

GTP, CTP and UTP as building units.

One DNA strand called the template strand serves as the matrix for the RNAsynthesis.

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.

Different types ofRNAPs

RNA Polymerase I is located in the nucleolus and transcribes ribosomalRNA (rRNA).

RNA Polymerase II is localized to the nucleus, and transcribes

messenger RNA (mRNA) and most small nuclear RNAs (snRNAs).


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RNA Polymerase III is localized to the nucleus (and possibly the

nucleolar- nucleoplasm interface), and transcribes tRNA and other small

RNAs.

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.

Rho-dependent termination uses a termination factor called factor

(rho factor) to stop RNA synthesis at specific sites.

This protein binds and runs along the mRNA towards the RNAP. When

-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 by which precursor messengerRNA is converted into mature messenger RNA (mRNA).

The three main modifications are

5' capping Addition of the 7 - Methylguanosine cap to 5 end is the

first step in pre-mRNA processing. This step occurs co-transcriptionally after

the growing RNA strand has reached 30 nucleotides. The process is catalyzedby a capping enzyme that associates with the carboxyl-terminal domain of RNA

polymerase II.

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 pre-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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Protein folding

Protein folding is the process by which a protein assumes its characteristicfunctional 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.

Most proteins can carry out their biological functions only when folding has

been completed, because three-dimensional shape of the proteins in the nativestate is critical to their function.

The process of folding in vivo 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.For example bacterial GroEL system which assists in the folding of globular

proteins.


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Posttranslational modification

Many proteins synthesised by translation are not functional as such. Manychanges takes place in the protein after synthesis which converts it into active

protein. These are known as post transcriptional modifications.

Trimming by Proteolytic Degradation

Many proteins are synthesised 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.

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.

Covalent Modifications

Proteins synthesised 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, citrullination, Acetylation etc


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The DNA sequence for a protein of interest canbe cloned or subcloned into a plasmidcontaining the lac promoter,

Then transformed into the bacterium E. coli.Addition of IPTG (a lactose analog) causes the

bacteria to express the protein of interest.However, this strategy does not always yieldfunctional protein, in which case, otherorganisms or tissue cultures may be moreeffective.

For example, Scerevisiae is often preferred tobacteria for proteins that undergo extensivepost translational modification. Nonetheless,bacterial expression has the advantage of easily

producing large amounts of protein.


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GENE EXPR SRS