Regulation of gene expression in eukaryotes

Dr. Namrata Chhabra Professor and Head

Department of BiochemistryS.S.R. Medical College, Mauritius

Biochemistry for medics- Lecture noteswww.namrata.co

1Biochemistry for medics-Lecture notes

http://www.namrata.co/



IntroductionIntroductionGene expression is the combined process of :o the transcription of a gene into mRNA, o the processing of that mRNA, and o its translation into protein (for protein-encoding

genes).


Levels of regulation of gene Levels of regulation of gene expressionexpression


Purpose of regulation of gene Purpose of regulation of gene expressionexpressionRegulated expression of genes is required for1) 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


Purpose of regulation of gene Purpose of regulation of gene expressionexpression2) Tissue specific differentiation and developmentThe genetic information present in each somatic cell of a

metazoan 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.


Control of gene ExpressionControl of gene Expression• Mammalian cells possess about 1000 times more

genetic information than does the bacterium Escherichia coli.

• Much of this additional genetic information is probably involved in regulation of gene expression during the differentiation of tissues and biologic processes in the multicellular organism and in ensuring that the organism can respond to complex environmental challenges.


Mechanism of regulation of gene Mechanism of regulation of gene expression- An overviewexpression- An overviewGene 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.


Mechanism of regulation of gene Mechanism of regulation of gene expression- An overviewexpression- An overviewTranscription control can result in tissue-specific

gene expression.In addition to transcription level controls, gene

expression can also be modulated byGene rearrangement, Gene amplification, Posttranscriptional modifications, and RNA stabilization.


Differences between gene Differences between gene expression in prokaryotes and expression in prokaryotes and eukaryoteseukaryotes

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

o The E. coli genome consists of a single, circular chromosome containing 4.6 Mb.

o This genome encodes approximately 2000 proteins.


1) Larger genome1) Larger genome• In comparison, the genome within a human cell

contains 23 pairs of chromosomes ranging in size from 50 to 250 Mb.

• Approximately 40,000 genes are present within the 3000 Mb of human DNA.

• It would be very difficult for a DNA-binding protein to recognize a unique site in this vast array of DNA sequences.

• More-elaborate mechanisms are required to achieve specificity


2) Different cell types2) Different cell typesDifferent 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.


3) Absence of operons3) Absence of operonsThe 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.


4) Chromatin structure4) Chromatin structureThe 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.


5) Uncoupled transcription and 5) Uncoupled transcription and translation processestranslation processes• In prokaryotes, transcription and translation are

coupled processes, the primary transcript is immediately translated.

• The transcription and translation are uncoupled in eukaryotes, eliminating some potential gene-regulatory mechanisms.

• The primary transcript in eukaryotes undergoes modifications to become a mature functional m RNA.


Mechanism of regulation of gene Mechanism of regulation of gene expression- Detailsexpression- Details1) Chromatin Remodeling• Chromatin structure provides an important level of

control of gene transcription. • With few exceptions, each cell contains the same

complement of genes (antibody-producing cells are a notable exception).

• The development of specialized organs, tissues, and cells and their function in the intact organism depend upon the differential expression of genes.

• Some of this differential expression is achieved by having different regions of chromatin available for transcription in cells from various tissues.


1) Chromatin Remodeling1) Chromatin Remodeling

Large regions of chromatin are transcriptionally inactive in some cells while they are either active or potentially active 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.



Formation and disruption of Formation and disruption of nucleosome structurenucleosome structure

• The presence of nucleosomes and of complexes of histones and DNA provide a barrier against the ready association of transcription factors with specific DNA regions.

• The disruption of nucleosome structure is therefore an important part of eukaryotic gene regulation and the processes involved are as follows-


Formation and disruption of Formation and disruption of nucleosome structure (contd.)nucleosome structure (contd.)i) Histone acetylation and deacetylation 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.


i) Histone Acetylation and i) Histone Acetylation and deacetylationdeacetylationThe amino-terminal tail

of histone H3 extends into a pocket in

which a lysine side chain can accept an acetyl group from acetyl CoA bound in an adjacent site.


Formation and disruption of Formation and disruption of nucleosome structure (contd.)nucleosome structure (contd.)ii) Modification of DNA Methylation of deoxycytidine residues in DNA may

effect gross changes in chromatin so as to preclude its active transcription.

Acute demethylation of deoxycytidine residues in a specific region of the tyrosine aminotransferase gene—in response to glucocorticoid hormones—has been associated with an increased rate of transcription of the gene.


Formation and disruption of Formation and disruption of nucleosome structure (contd.)nucleosome structure (contd.)iii) DNA binding proteinsThe binding of specific transcription factors to certain

DNA elements may result in disruption of nucleosomal structure.

Many eukaryotic genes have multiple protein-binding DNA elements.

The serial binding of transcription factors to these elements may either directly disrupt the structure of the nucleosome or prevent its re-formation.

These reactions result in chromatin-level structural changes that in the end increase DNA accessibility to other factors and the transcription machinery.


2) Enhancers and Repressors2) Enhancers and Repressors• Enhancer elements are DNA sequences, although

they have no promoter activity of their own but they greatly increase the activities of many promoters in eukaryotes.

• Enhancers function by serving as binding sites for specific regulatory proteins.

• An enhancer is effective only in the specific cell types in which appropriate regulatory proteins are expressed.


2) Enhancers and Repressors 2) Enhancers and Repressors (contd.)(contd.)• Enhancer elements can exert their

positive influence on transcription even when separated by thousands of base pairs from a promoter;

• they work when oriented in either direction; and they can work upstream (5') or downstream (3') from the promoter.

• Enhancers are promiscuous; they can stimulate any promoter in the vicinity and may act on more than one promoter.


2) Enhancers and Repressors 2) Enhancers and Repressors (contd.)(contd.)• The elements that decrease or repress the expression

of specific genes have also been identified. • Silencers are control regions of DNA that, like

enhancers, may be located thousands of base pairs away from the gene they control.

• However, when transcription factors bind to them, expression of the gene they control is repressed.

• Tissue-specific gene expression is mediated by enhancers or enhancer-like elements.


2) Enhancers and Repressors 2) Enhancers and Repressors (contd.)(contd.)


3) Locus control regions and 3) Locus control regions and InsulatorsInsulators• Some regions are controlled by complex DNA

elements called locus control regions (LCRs). • An LCR—with associated bound proteins—controls

the expression of a cluster of genes. The best-defined LCR regulates expression of the globin gene family over a large region of DNA.

• Another mechanism is provided by insulators. These DNA elements, also in association with one or more proteins, prevent an enhancer from acting on a promoter .


3) Locus control regions and 3) Locus control regions and InsulatorsInsulators

Biochemistry for medics-Lecture notes 28

4) Gene Amplification4) Gene AmplificationThe 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.


4) Gene Amplification (contd.)4) 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.


4) Gene Amplification(contd.)4) Gene Amplification(contd.)

Gene amplification has been demonstrated in patients receiving methotrexate for cancer.

The malignant cells can develop drug resistance by increasing the number of genes for dihydrofolate reductase, the target of Methotrexate.


5) Gene Rearrangement5) Gene RearrangementGene rearrangement is observed during

immunoglobulins synthesis. Immunoglobulins are composed of two polypeptides,

heavy (about 50 kDa) and light (about 25 kDa) chains. The mRNAs encoding these two protein subunits are

encoded by gene sequences that are subjected to extensive DNA sequence-coding changes.


5) Gene Rearrangement (contd.)5) Gene Rearrangement (contd.)

These DNA coding changes are needed for generating the required recognition diversity central to appropriate immune function.



The IgG light chain is composed of variable (VL), joining (JL), and constant (CL) domains or segments.

For particular subsets of IgG light chains, there are roughly 300 tandemly repeated VL gene coding segments, five tandemly arranged JL coding sequences, and roughly ten CL gene coding segments.

All of these multiple, distinct coding regions are located in the same region of the same chromosome.



However, a given functional IgG light chain transcription unit contains only the coding sequences for a single protein.

Thus, before a particular IgG light chain can be expressed, single VL, JL, and CL coding sequences must be recombined to generate a single, contiguous transcription unit excluding the multiple nonutilized segments.



This deletion of unused genetic information is accomplished by selective DNA recombination that removes the unwanted coding DNA while retaining the required coding sequences: one VL, one JL, and one CL sequence.


6) Alternative RNA Processing6) Alternative 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.


6) Alternative RNA Processing 6) Alternative RNA Processing (contd.) (contd.)

Alternative polyadenylation sites in the immunoglobulin (Ig M) heavy chain primary transcript result in mRNAs that are either 2700 bases long (m) or 2400 bases long (s).

This results in a different carboxyl terminal region of the encoded proteins such that the (m ) protein remains attached to the membrane of the B lymphocyte and the (s) immunoglobulin is secreted.


6) Alternative RNA Processing 6) Alternative RNA Processing (contd.) (contd.)


6) Alternative RNA Processing 6) Alternative RNA Processing (contd.)(contd.)

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


7) Class switching7) Class switchingIn 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.


7) Class switching (contd.)7) Class switching (contd.)It is produced by having two “Zeta” and two “Epsilon”

chains. By the sixth month of intrauterine life, embryonic Hb

is replaced by HbF consisting of “α2 and y2 chains. After birth HbF is replaced by adult type of Hb A

1(97%) and HbA2(3%). Thus the genes for a particular class of Hb are

switched off and for another class are switched on.


7) Class switching (contd.)7) Class switching (contd.)

Gene switching is also observed in the formation of immunoglobulins.

Ig M is the formed during primary immune response, while Ig G is formed during secondary immune response.


8) mRNA stability8) mRNA stability

Although most mRNAs in mammalian cells are very stable (half-lives measured in hours), some turn over very rapidly (half-lives of 10–30 minutes).

In certain instances, mRNA stability is subject to regulation.

This has important implications since there is usually a direct relationship between mRNA amount and the translation of that mRNA into its cognate protein.


8) mRNA stability (contd.)8) mRNA stability (contd.)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.


9)DNA binding proteins9)DNA binding proteins

Steroids such as estrogens bind to eukaryotic transcription factors called nuclear hormone receptors. These proteins are capable of binding DNA whether or not ligands are bound.

The binding of ligands induces a conformational change that allows the recruitment of additional proteins called co activators.


9) DNA binding proteins (contd.)9) DNA binding proteins (contd.)

Among the most important functions of co-activators is catalysis of the addition of acetyl groups to lysine residues in the tails of histone proteins.

Histone acetylation decreases the affinity of the histones for DNA, making additional genes accessible for transcription.


10)Specific motifs of regulatory 10)Specific motifs of regulatory proteinsproteinsCertain 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.


10) Specific motifs of regulatory 10) Specific motifs of regulatory proteins (contd.)proteins (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.


Three unique motifs of DNA Three unique motifs of DNA binding proteinsbinding proteins

Helix –turn- helixLeucine zipper

Zinc finger


11) RNA Editing11) RNA Editing

Enzyme- catalyzed deamination of a specific cytidine residue in the mRNA of apolipoprotein B-100 changes a codon for glutamine (CAA) to a stop codon (UAA).

Apolipoprotein B-48, a truncated version of the protein lacking the LDL receptor-binding domain, is generated by this posttranscriptional change in the mRNA sequence.


11)RNA Editing (contd.)11)RNA Editing (contd.)


SummarySummaryThe 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. 53Biochemistry for medics-Lecture notes

Education

Regulation of gene expression in eukaryotes