Regulation of Gene Expression Emphasizing Hormonal Action

Presented by: Sony Peter Lecturer

Department of BiochemistryGoba College of Medicine and Health Sciences

Madawalabu University

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

• Several steps in the gene expression process may be modulated, including the

• 1.transcription,• 2. RNA splicing• 3.translation, and • 4.post-translational modification of a protein.

• Process of alteration of gene expression been studied has been studied in in detail & involves modulation of gene transcription.

Transcription control can result in tissue specific gene expression& influenced by hormones, heavy metals etc.

In simple terms, regulation of gene expression is of two types1.positive regulation.2.negative regulation.

1. Positive Rregulation:When the expression of genetic information is

quantitatively increased by the presence of specific regulatory element, it is known as

positive regulation.

Elements or molecules modulating positive regulation is known as positive regulator.

2.Negative Regulation.

When the expression of genetic information is diminished by the presence of specific regulatory element, it is called the negative regulation.

The element or molecule mediating the negative regulation is called a negative regulator.

Human gene regulation occur at several levels

1. Regulation at the level of DNA2. Transcriptional control3. RNA Processing control4. RNA transport and localization control5. Translation control6. mRNA degradation control7. Protein activity control

Human gene regulation occur at several levels

1. Regulation at the level of DNA

The effect of chromosome structure on gene regulation.

DNA is wrapped tightly around histone proteins to form nucleosome.

Promoter blocking by nucleosome

• Histones positioned over promoter block the assembly of transcription factor complexes.

DNA Methylation

• Methylation of DNA was once thought to play a major role in gene regulation.

• Many inactive human genes are methylated.• Methylation is now viewed as having a less

direct role, blocking accidental transcription of “Turned Off Genes”.

• Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation.

2. Transcriptional Control

Regulating Promoter AccessOne way to control transcription is to regulate the initiation of transcription.

Protein- binding nucleotide sequences on the DNA regulate the initiation of transcription by modulating the ability of RNA polymerase to bind to the promoter.

Binding the protein to the regulatory sequence either blocks transcription by getting in the way of

RNA polymerase, or stimulate transcription by facilitating the binding of RNA polymerase to the

Promoter

Transcription control in Prokaryotes

Significance:In Bacteria, the primary function of gene control is to adjust the cell’s activities to its immediate environment.Changes in gene expression alter which enzymes are present in the cell in response to the quantity and type of available nutrients and the amount of Oxygen present.Almost all of these changes are fully reversible, allowing the cell to adjust its enzyme levels up or down as the environment changes.

Transcriptional Control in Eukaryotes

• In human, with relatively constant internal environment, the primary function of gene control in a cell is not to respond to that cell’s immediate environment, but rather to participate in regulating the body as a whole.

• Transcription control, more common, is effected by binding of protein to regulatory sequences within the DNA.

Regulatory proteins read DNA without unwinding it.

• Within the major groove, the nucleotides hydrophobic methyl groups, hydrogen atoms and hydrogen bond donors and acceptors protrude.

• The pattern created by these chemical groups is unique for each of the four possible base-pair arrangement, providing a ready way for a protein nestled in the groove to read the sequence of base

Major groove and Minor groove of DNA

CH3- Hydrophobic methyl groupYellow color- Hydrogen bond donorsRed color- Hydrogen bond acceptorsBlue color- Hydrogen atoms unable to form hydrogen bonds

DNA Binding Motifs

• DNA binding motif on the protein chain permit it to interlock with the major groove of the DNA helix.

• 1. The Helix-Turn-Helix motif• 2. Homeodomain motif• 3. The Zinc Finger motif• 4. The Leucine Zipper motif

1. The Helix-Turn-Helix motif

This motif is constructed from two alpha-helical segments of the protein linked by a short non helical segment, the turn.

This motif has been identified in hundreds of DNA- binding proteins.

2. Homeodomain motif

It is a special class of Helix-Turn-Helix motif. More than 50 of these regulatory proteins have been analyzed and they all contain a nearly identical sequence of 60 amino acids, the homeodomain.

3. The Zinc Finger motif

• This uses one or more zinc atoms to coordinate its binding to DNA called zinc fingers.

• The more zinc fingers in the cluster, the stronger the protein binds to the DNA.

4. The Leucine Zipper motif

• Here two different protein subunits cooperate to create a single DNA binding site.

• This motif is created where a region on one of the subunits containing several hydrophobic amino acids (usually Leucine) interacts with a similar regions on the other subunit.

The DNA Binding motifs

Transcriptional control in humans operates at a distance

• In human, many genes must interact with one another.

• Many regulatory sequences scattered around the chromosome can influence a particular gene’s transcription

The transcription complex that positions RNA polymerase at the beginning of a gene consists of 4 kinds of proteins.• Basal factors• Co-activators• Activators• Repressor

Human transcription complex

RNA Processing

Processing of the primary transcriptThis include excision of introns, capping of 5’ end and addition of Polyadenylate tail to 3’ end of the primary transcript.

1. Exons and Introns- Introns removed by RNA Processing or RNA SplicingSmall nuclear ribonucleoprotein (snRNPs) play a role in RNA splicing.

These particles reside in the nucleus and composed of proteins and a special type of RNA called small nuclear RNA (snRNA).

Multiple snRNPs base pair with introns to form a larger complex called Spliceosome. The introns loops out and is exised.

How spliceosomes process RNA

RNA processing cont.…….

RNA splicing provides a potential point where the expression of a gene can be controlled, because exons can be spliced together in different ways, allowing a variety of different polypeptides to be assembled from the same gene.

In many cases, gene expression is regulated by changing which splicing event occur during different stages of development or in different tissues.

Example, The alternative splicing in action found in thyroid and hypothalamus.Thyroid produce CalcitoninHypothalamus produce CGRP (Calcitonin gene related peptide) as a part of their function.Different purpose, but the hormones are made using the same transcript.

RNA processing cont.…….

RNA processing cont.…….

• 7-methylguanine capping at 5’ endThis capping at 5’ end of the transcript is appended by nucleoside phosphohydrolase, guanyltransferase and guanine methylferase.

• 3’polyadenylationAfter the 5’ capping, the 3’ end of the transcript will be added by a long poly A tail, consisting of a string of about 20-200 adenine ribonucleotides byMg2+ dependent nucleoplasmic poly-A polymerase.

RNA transport and localization control

Processed mRNA transcript exit the nucleus through nuclear pores.Its active process. Transcript has to be recognized by the receptors lining the interior of the pore. Poly A tail play a role in this recognition

Translation control

• The translation of processed mRNA transcript by the ribosomes in the cytoplasm involves a complex of proteins called Translation factors.

• In some cases, gene expression is regulated by modification of one or more of these factors.

• In other instances, Translation repressor proteins shut down translation by binding to the beginning of the transcript, so that it cannot attach to the ribosome.

• Example, the production of ferritin is normally shut off by a translation repressor protein called aconitase.

mRNA degradation control

• Another aspect that affects gene expression is the stability of mRNA transcripts in the cell cytoplasm.

• Human mRNA transcripts are very stable compared to lower organisms.• Example, beta-globin gene transcript have a half-life of over 10 hrs. However, the transcripts encoding regulatory proteins and growth factors are usually much less stable, with a half-life < 1 hr.

WHAT MAKES THESE PARTICULAR TRANSCRIPTS SO UNSTABLE?

- A sequence of A and U nucleotides near 3’ end- Destabilize and prone to enzyme attack- Some mRNA transcripts digested by endonucleases.

mRNA degradation control con……

Example, Histone transcripts have a half-life of about 1 hr in the cells that are actively synthesizing DNA, at other times during cell cycle, the poly A tail is lost and the transcripts are degraded within minutes.

The short half-lives of the mRNA transcripts of many regulatory genes are critical to the function of those genes, as they enable the levels of regulatory proteins in the cell to be altered rapidly.

• Some hormones which enhance the production of proteins also increase the half life of the protein’s mRNA.

Estrogen : t1/2 from 2- 5hr to >24hr

Prolactin : t1/2 from 5 hr to 92hr

Protein activity control

• Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision.

• Proteins builded after translation can be• • functional or• have to undergo a maturation process (exo/- endopeptidation) or functional group addition by phosphorylation, acetylation,methylation … to functional.

Hormonal control of gene expression

Hormones are molecules that are produced in one cellular location in the human body and whose effects are seen in another tissue or cell type.

Hormones can be proteins or steroidsEg, insulin, epinephrine estrogen, progesterone, testosterone control gene expression.

The protein hormones do not enter the cell, but bind to receptors in the cell membrane and mediate gene expression through intermediate molecules.

The steroid hormones enter the cell and interact with steroid receptor proteins to control gene expression.

Peptide hormone action

Peptide hormone action con…..

Hormones are synthesized in various specialized secretory cells (endocrine cells) and are released into blood stream. The peptide hormones do not normally enter the cells because of their relative large size. Their effect is mediated by receptor proteins located in target cell membranes and by the intracellular level of cAMP (called the secondary messenger).The cAMP activates a protein kinase (A- Kinase) which phosphorylates (activates) many specific enzymes.

Steroid hormone action

Steroid hormone action con…….

Steroid hormones are small molecules that readily enter cells through plasma membrane.Once inside the appropriate target cells, the steroid hormones become tightly bound to specific receptor proteins which are present only in the cytoplasm of target cells.The hormone-receptor protein complex activate the transcription of specific genes according to following two methods:1. The H-R PC interact with specific non-histone chromosome proteins and

this interaction stimulates the transcription of correct genes.2. H-R PC activate transcription of target genes by binding to specific DNA

sequences present in the cis-acting regulatory regions of the genes.

In both of these cases, the H-R PC would function as positive regulators or activators of transcription.

Steroid hormone action con…….

Glucocorticoid hormones influence nutrient metabolism in most of the body cells by promoting the metabolism of glucose, proteins and fats.

The effect of glucocorticoids is to activate the transcription of specific genes.The hormone is released from the endocrine cells and secreted into the blood stream when the individual is fasting and needs to regulate its blood levels of glucose, a.a- and fats.

The hormone molecules diffuse across the plasma membrane of target cells and bind to glucocorticoid receptors.

Steroid hormone action con…….

Steroid hormone action con…….

The thyroid hormones tri iodo thyronine and tetra iodo thyronine, T3 and T4 have marked effect on the growth, development and metabolic function of virtually all organ systems and tissues of human body.

A significant amount of T3 is derived from T4 by 5’ deiodinase in various tissues.Because of the marked alteration in the rate of oxygen consumption identified in hypothyroidism and hyperthyroidism, initial studies on mechanism of action of the thyroid hormones focused on mitochondrial function. Experimental works shows that T-H administration increased the rate of RNA synthesis, specially stimulating the accumulation of mRNAs which codes for specific proteins.

Steroid hormone action con…….

The following T-H depended effects have also been analyzed:

1. Stimulation and regulation of growth hormone gene expression2. Stimulation of malic enzyme mRNA in the liver3. Stimulation of several other genes that encode hepatic proteins of

unknown function.4. Stimulation of the alpha-myosin heavy chain gene in the myocardium.5. Inhibition of thyrotrophin and the beta-myosin heavy chain gene.