Regulation of Gene ExpressionEmphasizing Hormonal Action Presented by: Sony Peter Lecturer Department of Biochemistry Goba 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 types 1.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 isdiminished by the presence of specificregulatory element, it is called the negativeregulation.The element or molecule mediating thenegative regulation is called a negativeregulator.
Human gene regulation occur at several levels1. Regulation at the level of DNA2. Transcriptional control3. RNA Processing control4. RNA transport and localization control5. Translation control6. mRNA degradation control7. Protein activity control
1. Regulation at the levelof DNAThe effect of chromosomestructure on generegulation.DNA is wrapped tightlyaround histone proteins toform 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 ControlRegulating Promoter AccessOne way to control transcription is to regulatethe initiation of transcription.Protein- binding nucleotide sequences on theDNA regulate the initiation of transcription bymodulating the ability of RNA polymerase tobind to the promoter.
Binding the protein to the regulatory sequenceeither 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 ProkaryotesSignificance:In Bacteria, the primary function of gene control isto adjust the cell’s activities to its immediateenvironment.Changes in gene expression alter which enzymesare present in the cell in response to the quantityand type of available nutrients and the amount ofOxygen present.Almost all of these changes are fully reversible,allowing the cell to adjust its enzyme levels up ordown 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 DNACH3- 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 motifThis motif is constructed from two alpha-helicalsegments of the protein linked by a short nonhelical segment, the turn.This motif has been identified in hundreds ofDNA- binding proteins.
2. Homeodomain motifIt is a special class of Helix-Turn-Helix motif.More than 50 of these regulatory proteins havebeen analyzed and they all contain a nearlyidentical sequence of 60 amino acids, thehomeodomain.
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.
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 thatpositions RNA polymerase at thebeginning of a gene consists of 4kinds of proteins.• Basal factors• Co-activators• Activators• Repressor
RNA ProcessingProcessing of the primary transcriptThis include excision of introns, capping of 5’ end and addition ofPolyadenylate 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 specialtype of RNA called small nuclear RNA (snRNA).Multiple snRNPs base pair with introns to form a larger complex calledSpliceosome. The introns loops out and is exised.
RNA processing cont.…….RNA splicing provides a potential point where the expression of a gene can becontrolled, because exons can be spliced together in different ways, allowinga variety of different polypeptides to be assembled from the same gene.In many cases, gene expression is regulated by changing which splicing eventoccur during different stages of development or in different tissues.Example, The alternative splicing in action found in thyroid andhypothalamus.Thyroid produce CalcitoninHypothalamus produce CGRP (Calcitonin gene related peptide) as a part oftheir function.Different purpose, but the hormones are made using the same transcript.
RNA processing cont.…….• 7-methylguanine capping at 5’ endThis capping at 5’ end of the transcript is appended bynucleoside phosphohydrolase, guanyltransferase andguanine methylferase.• 3’polyadenylationAfter the 5’ capping, the 3’ end of the transcript will beadded by a long poly A tail, consisting of a string ofabout 20-200 adenine ribonucleotides byMg2+dependent nucleoplasmic poly-A polymerase.
RNA transport and localization controlProcessed mRNA transcript exit the nucleus through nuclear pores.Its active process.Transcript has to be recognized by the receptors lining the interior of thepore. 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 areusually 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 areactively synthesizing DNA, at other times during cell cycle, the poly A tail islost and the transcripts are degraded within minutes.• 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 92hrThe short half-lives of the mRNA transcripts of many regulatory genes arecritical to the function of those genes, as they enable the levels of regulatoryproteins in the cell to be altered rapidly.
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 expressionHormones are molecules that are produced in one cellular location in thehuman body and whose effects are seen in another tissue or cell type.Hormones can be proteins or steroidsEg, insulin, epinephrine estrogen, progesterone, testosterone control geneexpression.The protein hormones do not enter the cell, but bind to receptors in the cellmembrane and mediate gene expression through intermediate molecules.The steroid hormones enter the cell and interact with steroid receptorproteins to control gene expression.
Peptide hormone action con…..Hormones are synthesized in various specialized secretory cells (endocrinecells) and are released into blood stream. The peptide hormones do not normally enter the cells because of theirrelative large size. Their effect is mediated by receptor proteins located intarget cell membranes and by the intracellular level of cAMP (called thesecondary messenger).The cAMP activates a protein kinase (A- Kinase) which phosphorylates(activates) many specific enzymes.
Steroid hormone action con…….Steroid hormones are small molecules that readily enter cells through plasmamembrane.Once inside the appropriate target cells, the steroid hormones become tightlybound to specific receptor proteins which are present only in the cytoplasmof target cells.The hormone-receptor protein complex activate the transcription of specificgenes 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 oractivators of transcription.
Steroid hormone action con…….Glucocorticoid hormones influence nutrient metabolism in most of the bodycells 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 bloodstream when the individual is fasting and needs to regulate its blood levels ofglucose, a.a- and fats.The hormone molecules diffuse across the plasma membrane of target cellsand bind to glucocorticoid receptors.
Steroid hormone action con…….The thyroid hormones tri iodo thyronine and tetra iodo thyronine, T3 and T4have marked effect on the growth, development and metabolic function ofvirtually all organ systems and tissues of human body.A significant amount of T3 is derived from T4 by 5’ deiodinase in varioustissues.Because of the marked alteration in the rate of oxygen consumptionidentified in hypothyroidism and hyperthyroidism, initial studies onmechanism of action of the thyroid hormones focused on mitochondrialfunction.Experimental works shows that T-H administration increased the rate of RNAsynthesis, specially stimulating the accumulation of mRNAs which codes forspecific 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.