Gene expression,Regulation of gene expression by dr.Tasnim

Gene Expression,Regulation of gene Expression,Human genom project

DR.TASNIM ARA JHILKYDR.TASNIM ARA JHILKY

MD PART IIMD PART II

Phase APhase A

DEPARTMENT OF BIOCHEMISTRYDEPARTMENT OF BIOCHEMISTRY

SIR SALIMULLAH MEDICAL COLLEGE MIDFORD,DHAKASIR SALIMULLAH MEDICAL COLLEGE MIDFORD,DHAKA

1.

Gene Expression

• It is the process by which information from a gene is used in the synthesis of a functional gene product.

• These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA.

http://www.news-medical.net/health/What-is-RNA.aspx

Gene expression regulation:

Both of these cells contain the same

genome, but they express different RNAs

and proteins.

Classification of gene with respect to their Expression:

• Constitutive ( house keeping) genes:• 1- Are expressed at a fixed rate, irrespective to the

cell condition.• 2- Their structure is simpler• Controllable genes: • 1- Are expressed only as needed. Their amount may

increase or decrease with respect to their basal level in different condition.

• 2- Their structure is relatively complicated with some response elements

Levels of regulation of gene Levels of regulation of gene expressionexpression

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1.positive regulation:

• When the expression of genetic is quantitatively increased by the presence of specific regulatory

element is known as positive regulation.

• Element modulating positive regulation is known as activator or positive regulator.

2.Negative regulation.

•when the expression of genetic information diminished by the presence of specific regulatory element.

•The element or molecule mediating the negative regulation is said to be repressor.

Purpose of regulation of gene Purpose of regulation of gene expressionexpression

Regulated expression of genes is required for

1) Adaptation- Cells of multicellular organisms respond to varying conditions.

Such cells exposed to hormones and growth factors change substantially in –

o shape, o growth rate, and o other characteristics

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Purpose of regulation of gene Purpose of regulation of gene expressionexpression

2) Tissue specific differentiation and developmentThe genetic information present in each somatic cell of

a organism is practically identical. Cells from muscle and nerve tissue show strikingly

different morphologies and other properties, yet they contain exactly the same DNA.

These diverse properties are the result of differences in gene expression.

Expression of the genetic information is regulated during ontogeny and differentiation of the organism and its cellular components.

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Mechanism of regulation of gene Mechanism of regulation of gene expression-an overviewexpression-an overview

• Gene activity is controlled first and foremost at the level of transcription.

• Much of this control is achieved through the interplay between proteins that bind to specific DNA sequences and their DNA binding sites.

• This can have a positive or negative effect on transcription.

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Mechanism of regulation of gene Mechanism of regulation of gene expression- An overviewexpression- An overview

• Transcription control can result in tissue-specific gene expression.

• In addition to transcription level controls, gene expression can also be modulated by

• Gene rearrangement, • Gene amplification, • Posttranscriptional modifications, and • RNA stabilization.

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Differences between gene expression Differences between gene expression in prokaryotes and eukaryotesin prokaryotes and eukaryotes

Gene regulation is significantly more complex in eukaryotes than in prokaryotes for a number of reasons:

1) First, the genome being regulated is significantly larger

• Human cell have larger genome

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2) Different cell types2) Different cell types

• Different cell types are present in most eukaryotes.

• Liver and pancreatic cells, for example, differ dramatically in the genes that are highly expressed.

• Different mechanisms are involved in the regulation of such genes.

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3) Absence of operons3) Absence of operons

• The eukaryotic genes are not generally organized into operons as are there in prokaryotes

• Instead, genes that encode proteins for steps within a given pathway are often spread widely across the genome.

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4) Chromatin structure4) Chromatin structure

• The DNA in eukaryotic cells is extensively folded and packed into the protein-DNA complex called chromatin.

• Histones are an important part of this complex since they both form the structures known as nucleosomes and also contribute significantly into gene regulatory mechanisms.

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Principles of Gene Regulation in prokaryote

Most prokaryotic genes are regulated in unitscalled operons.Francois Jacob & Jacques Monod, 1961.This is largely based on regulation of lactose metabolism. By intestinal bact. E.coli.

Different ways for regulation of gene expression in bacteria:

• 1- Promoter recognition.• 2-Transcription elongation( Attenuation).

Lac Operon – Lac Operon – • Bacteria such as E. coli usually rely on glucose

as their source of carbon and energy.• However, when glucose is scarce, E. coli can

use lactose as their carbon source even though this disaccharide does not lie on any major metabolic pathways.

• An essential enzyme in the metabolism of lactose is β-galactosidase, which hydrolyzes lactose into galactose and glucose

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Components of Lac OperonComponents of Lac Operon

• The genetic elements of the model are a regulator gene, a regulatory DNA sequence called an operator site, and a set of structural genes.

• The regulator gene encodes a repressor protein that binds to the operator site.

• The binding of the repressor to the operator prevents transcription of the structural genes.

• If the operator is not bound by a repressor molecules ,RNA polymeraze passes over the operator and reaches the protein coding genes which is transcribes to mRNA06/05/17 20.

Components of Lac OperonComponents of Lac Operon• For the lactose (lac) Operon, the i gene encodes the

repressor, o is the operator site, and the z, y, and a genes are the structural genes for β -galactosidase, the permease, and the transacetylase, respectively.

• The Operon also contains a promoter region (denoted by p), where RNA polymerase binds and two additional sites operator and CAP site is bound by cAMP and catabolite activator protein.

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06/05/17 22Biochemistry For Medics

•The z, y, and a genes are transcribed to give a single mRNA molecule that encodes all three proteins.• An mRNA molecule encoding more than one protein is known as a polygenic or polycistronic



Regulation of Expression of Lac OperonRegulation of Expression of Lac Operon1) In the absence of lactose/When glucose is available- Lac

Operon remains repressed (turned off)due to the presence of lac repressor at the operator site- (Negative control).

2) In the presence of only Lactose- Lac Operon is turned on. the structural genes are transcribed and the lactose metabolizing enzymes are synthesized .A small amount of lactose is converted to allolactose and binds with repressor protein and change its conformation ,so repressor can not binds with operator

In the absence of glucose adenylyl cyclase active ,made cyclic AMP which binds with CAP causing RNA polymerase to more efficiently initiate transcription.06/05/17 25.

Regulation of Expression of Lac OperonRegulation of Expression of Lac Operon

3) In the presence of both glucose and lactose- CAP -cAMP complex is not formed(due to inhibition of adenylyl cyclase),RNA polymerase can not initiate the transcription of structural genes despite the fact that the operator site is vacant due to the binding of lactose/allolactose with lac repressor. Lac Operon remains in the repressed state.

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Maximum Expression of Lac OperonMaximum Expression of Lac OperonThe lac Operon is controlled by two distinct DNA binding factors; One that acts positively (cAMP-CRP complex) andThe other that acts negatively (LacI repressor). Maximal activity of the lac Operon occurs when glucose levels are low (high cAMP with CAP activation) and lactose is presen,t LacI is prevented from binding to the operator).06/05/17 27.

Lac IPromoter

geneOperator

geneLac Z Lac Y Lac A

R

Translation & Transcription

RNA polymerase

No Gene Expression

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.

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Combinatory Regulation of Lac Operon:CAP: catabolite activator protein; breakdown of lactose when glucose is low and lactose is present

……..

• The genetic constitutions of nearly all metazoan somatic cells are identical.

• Tissue or cell specificity is dictated by differences in gene expression of this complement of genes.

• Alterations in gene expression allow a cell to adapt to environmental changes.

• Gene expression can be controlled at multiple levels by chromatin modifications ,changes in transcription, RNA processing, localization, and stability or utilization.

• Gene amplification and rearrangements also influence gene expression. 33.

Eukaryotic gene regulation occurs at several levels:

Changes in DNA level

• Is done by • Gene amplification• Gene rearrangement

Gene AmplificationGene Amplification

• The gene product can be increased by increasing the number of genes available for transcription of specific molecules

• Among the repetitive DNA sequences are hundreds of copies of ribosomal RNA genes and tRNA genes.

• During early development of metazoans, there is an abrupt increase in the need for ribosomal RNA and messenger RNA molecules for proteins that make up such organs as the eggshell.

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Gene Amplification (contd.)Gene Amplification (contd.)• Such requirements are fulfilled by amplification of

these specific genes.• Subsequently, these amplified genes, presumably

generated by a process of repeated initiations during DNA synthesis, provide multiple sites for gene transcription.

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5) Gene Rearrangement 5) Gene Rearrangement Gene rearrangement is observed during immunoglobulins synthesis.

here segment of DNA move from one location to another in the genom.These DNA coding changes are needed for generating the required recognition diversity central to appropriate immune function.

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Mechanism of regulation of gene Mechanism of regulation of gene expression-Transcriptional level expression-Transcriptional level 1) 1) Chromatin RemodelingChromatin Remodeling

• Large regions of chromatin are transcriptionally inactive /heterochromatin in some cells while they are either active or potentially active /euchromatin in other specialized cells

• For example, the DNA containing the -globin gene cluster is in "active" chromatin in the reticulocytes but in "inactive" chromatin in muscle cells.

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40Biochemistry for medics-Lecture notes

Cont……

Use of alternative promoter

Some genes has series of promoter s showing tissue specific expression.so in different tissue different transcript and different protein produced

sSSlternative promoter•S

Acetylation is known to occur on lysine residues in the amino terminal tails of histone molecules.

This modification reduces the positive charge of these tails and decreases the binding affinity of histone for the negatively charged DNA.

Accordingly, the acetylation of histones could result in disruption of nucleosomal structure and allow readier access of transcription factors to cognate regulatory DNA elements.

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2)Histone acetylation and deacetylation

3)DNA methylation:is the addition or removal of a methyl group predominantely

where cytosine bases occur consecutively.make bases occur consecutively.

Con….

• Individual gene regulation• Negative regulation• Positive regulation

Post transcriptional regulation of gene expresstion

• Done by• Alternative splicing• Class switching• Regulation of RNA stability• RNA editing

Alternative RNA ProcessingAlternative RNA Processing

• Eukaryotic cells also employ alternative RNA processing to control gene expression.

• This can result when alternative promoters, intron-exon splice sites, or polyadenylation sites are used.

• Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differently in different tissues.

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Alternative RNA Processing (contd.)Alternative RNA Processing (contd.)

Alternative splicing and processing, results in the formation of seven unique -tropomyosin mRNAs in seven different tissues.


Calcitonin gene-related peptide

61

cell 1

cell 2

(four exons)1 2 3 4

1, 2 & 3 1, 2 & 4

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

Class switchingClass switching

In this process one gene is switched off and a closely related gene takes up the function.

During intrauterine life embryonic Hb is the first Hb to be formed.

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mRNA stability mRNA stability • Changes in the stability of a specific mRNA can

therefore have major effects on biologic processes.• The stability of the m RNA can be influenced by

hormones and certain other effectors.• The ends of mRNA molecules are involved in

mRNA stability. • The 5' cap structure in eukaryotic mRNA prevents

attack by 5' exonucleases, and the poly(A) tail prohibits the action of 3' exonucleases.

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RNA Editing RNA Editing


Translational regulation

• Regulating the activity of initiation factors ,particularly initiation factor-2

• Action of IF2 is inhibited when phosphorylated,so by phosphprylation and prevention of phosphorylation of IF2,translation can be regulated.

Post translational regulation

• Protein activation

Some proteins are not active when first formed.so to be active they undergo modifications

Folding

Proteolysis

Engymatic cleavage

Regulation of protein degradation or turnover.

Specific motifs of regulatory proteinsSpecific motifs of regulatory proteins

• Certain DNA binding proteins having specific motifs bind certain region of DNA to influence the rate of transcription.

• The specificity involved in the control of transcription requires that regulatory proteins bind with high affinity to the correct region of DNA.

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Specific motifs of regulatory proteins Specific motifs of regulatory proteins (contd.)(contd.)

• Three unique motifs—the helix-turn-helix, the zinc finger, and the leucine zipper—account for many of these specific protein-DNA interactions.

• The motifs found in these proteins are unique; their presence in a protein of unknown function suggests that the protein may bind to DNA.

• The protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces.

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Three unique motifs of DNA binding Three unique motifs of DNA binding proteinsproteins

Helix –turn- helixLeucine zipper

Zinc finger


Method of quantification of gene expression

• 1)detection of mRNAlevel by• Northern blotting• Microarrays• Reverse transcriptase PCR• 2)Analysis of protein level by• ELISA• Western blotting• Photometry• proteomics

HUMAN GENOME HUMAN GENOME PROJECTPROJECT

The Human GenomeThe Human Genome• The human genome is the complete set of genetic

information for humans (Homo sapiens).• The human genome is by far the most complex and largest

genome. • Its size spans a length of about 6 feet of DNA, containing

more than 30,000 genes.• The DNA material is organized into a haploid chromosomal

set of 22 (autosome) and one sex chromosome (X or Y).

Male Female

Human Genome Sequencing 2/11/200122 autosome + 2 sex chromosomes

From NCBI

What was Human Genome Project(HGP)What was Human Genome Project(HGP)

• The Human Genome Project was an international research effort to determine the sequence of the human genome and identify the genes that it contains.

• The US Human Genome Project is a 13 year effort, which is coordinated by the

– Department of Energy (DOE) and– National Institutes of Health

(NIH).

1.To identify all the genes in human DNA.2.To develop a genetic linkage map of human genome.3.To obtain a physical map of human genome.

4.To develop technology for the management of human genome information.

5.To know the function of genes.6.Determine the sequences of the 3 billion chemical

base pairs that make up human DNA.7.Store this information in public databases.8.Develop tools for data analysis.9.Transfer related technologies to the private sectors.

Goals of Human Genome ProjectGoals of Human Genome Project

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Gene expression,Regulation of gene expression by dr.Tasnim

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