Regulation of gene expression Kamila Balušíková

Gene expression vs. Regulation of gene expressionGene expression vs. Regulation of gene expression Unicellular organisms: requirements for adaptation to changed environmental conditions. Multicellular organisms: requirements for the selective expression of genes → relevant differentiation status of various cell types ► cell differentiation arises because cells make and accumulate different sets of RNA and protein molecules; that is, they express different genes

Whether a gene is expressed or not depends on a variety of factors:  the type of cell  its surroundings  its age  extracellular signals

 each cell contains complete genome but express only part of the genes  THE NEED OF REGULATION  cells have to be able to respond to changes in environment  THE NEED OF REGULATION

(B)

Levels of the regulation of GE Genome (DNA) Transcription (DNA → RNA/primary transcript) – transcriptional control Posttranscriptional modifications (RNA/primary transcript → mRNA) – processing control, RNA transport and localization control Translation (mRNA → polypeptide chain) – translational control, mRNA degradation control Posttranslational modifications (polypeptide chain → functional protein) Protein degradation (functional protein → decomposed protein) – protein activity control

Regulation of GE Regulation of transcription – most economic X Regulation of protein activity – fastest

Genome (DNA) Regulation of the access to genomic DNA –Condensation/decondensation of the chromosome the higher level of DNA condensation the less DNA accessibility for transcription factors and RNA polymerases –Structure of chromatin (heterochromatin x euchromatin

X inactivation  X inactivation (always only 1 X chromosome is active - Xa, Xi)  Non-translated (non-coding) RNA – Xist (X-inactivation specific transcript), Xi is coated by Xist RNA which is transcribed by this Xi → if the XIC is spread over the entire chr. – inactivation of this chr.)  Limiting blocking factor – binds to X inactivation center (XIC) and it blocks inactivation (only in 1 Xa)  Xa – no coating by Xist, no inactivation

Position effect (PEV – position effect variegation) Each chromosome (and gene) has its own place in nucleus (sufficient amount of RNA polymerase, regulation factors, euchromatin) Difference in the gene expression which depends on the position of his gene in genome = position effect Experiment with drosofila: white gene (color of the eye) if moved near the heterochromatin – inactivation of the gene)

Acetylation of histones Histone acetylation – histon acetylase → activation Histone deacetylase removes the acetyl group from histone → DNA less accessible

Methylation Genes which are not expressed can be methylated (→ their expression is blocked) Enzyme methylase - catalyzes the methylation of cytosine in DNA (5-methylcytosine) Methylation of CG sites (sequence: -C*G-p-C*G-) The degree of DNA methylation corresponds to the degree of gene expression (methzlated gene = non-expressed gene) Genomic imprinting – only one copy of gene (maternal x paternal) is active

Epigenetics changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence Epigenetics processes are usually caused by transcription repression which is controlled by chromatin modulation –Genomic imprinting –Position effect –Gene dose compensation (X inactivation) –Chemical modification of DNA, histones –Chromatin remodeling

Transcription Controlled by the binding of specific regulatory proteins to specific regulatory DNA sequences - Based on protein-DNA interaction (DNA binding proteins) Most economic way of regulation Eucaryotic cells: each gene under individual control, complex regulation Procaryotic cells: regulation of transcription of whole operon, simple regulation Repressors/activators bind to specific promoter sequences

DNA binding proteins recognize sequences, usually bind to the major groove

Regulatory DNA sequences – are needed in order to switch the gene on or off procaryotes – 10 nucleotide pairs short (simple gene switches) eucaryotes – nucleotide pairs away (microprocessors) Gene regulatory proteins –bind to regulatory DNA sequences  The combination of a DNA sequence and its associated protein molecules acts as the switch to control transcription  single contact = a non-covalent contact between one base pair and amino acid  Regulatory protein contains such specific contacts  DNA - protein motifs = general folding patterns of regulatory proteins

DNA - protein motifs = general folding patterns of regulatory proteins Types of DNA binding proteins: 1.Leucine zipper 2.Helix-loop-helix 3.Helix-turn-helix 4.Zinc finger

Zinc finger Zn atom - stabilization type helix + β sheet helix + helix (dimerization) Helix-loop-helix Longer loop – position of helix is not fixed Dimerization (homo and heterodimer) Regulation – dimerization with shorter protein

Leucine zipper Dimerization (hydrofobic interaction of leucins) of α helixes Helix-turn-helix Short loop, fixed angle C terminal helix: binding to major groove

Procaryotes effective response to quickly changing physical /chemical conditions of environment main purpose: survival of the cell regulation especially on the transcription level very short lifetime of mRNA (cca 3 min.) polygenic mRNA Regulation of gene expression best studied in proteins with enzymatic function

Regulation of transcription in procaryotes OPERON – transcription unit, a cluster of genes on the chromosome, which are regulated by a single promoter and operator, they are transcribed as one long mRNA molecule –1 mRNA (with several genes) = 1 transcription unit –polycistronic transcript PROMOTER – initiation site, where transcription actually begins, upstream region, a sequence which contains sites that are required for the RNA polymerase to bind to the promoter

OPERATOR – a short DNA sequence (cca 15 nucleotides in length) within the promoter which is recognized by a gene regulatory proteins regulatory gene – is localized outside the operon, codes for regulatory protein, its expression is usually constitutive and controlled by its own promoter regulatory proteins – bind to the promoter/operator, (encoded by regulatory gene) –Repressor protein - switches genes off, represses them (synthesis of tryptophan) –Activator protein - switches genes on, activates them (degradation of sugars, CAP)

Regulation of transcription in procaryotes Simple regulation Specific sigma factors (interaction with RNA polymerase) Regulatory proteins: activators, repressors

Trp operon Tryptophan repressor

Regulation of transcription in eucaryotes Eucaryotes x procaryotes RNA-polymerase → procaryotes 1 x eucaryotes 3 RNA pol I (rRNA), II (mRNA), III (tRNA, small RNA) general transcription factorsEucaryotic RNA-polymerase needs a large set of proteins - general transcription factors - for initiation of transcription (they assemble at the promoter (TATA box), they are highly universal, evolutionary conserved)

specific transcription factorsEucaryotes can influence the initiation of transcription by specific transcription factors (repressors and activators) even when they are bound to DNA thousands of nucleotide pairs away from the promoter, this feature allows a single promoter to be controlled by an almost unlimited number of regulatory sequences scattered along the DNA the DNA, at this time, loops out to allow all proteins to come into contact, regulation is complex - combinatorial control Eucaryotic transcription initiation must take account of the packing of DNA into nucleosomes and more compact forms of chromatin structure

TATA box is highly conserved promoter in eucaryotes

Preinitiation complex and initiation of transcription RNA polymerase II preinitiation complex presence of general transcription factors (TFII) are needed for assembly of Pol II at the DNA TFII create multimers and highly conserved Proteins (TFII) are bound in specific order and create RNA pol II preinitiation complex

TBP binds to TATA-box and folds DNA double helix TBP is subunit of TFIID

Initiation of transcription

General transcription factors are usually not sufficient, the presence of activator proteins is needed ( they help to assemble general transcription factors and RNA pol. into initiation complex ) x repressors slow down, block this process enhancer – binding site for activator prot ein

Complex of regulatory proteins – combination of the effect of several regulatory proteins

Combinatorial control groups of proteins work together to determine the expression of a single gene The effects of multiple gene regulatory proteins combine to determine the rate of transcription initiation The effect of a single gene regulatory protein can be decisive in switching any particular gene on or off This do so by completing the combination

Activators Often in distant regions Synergic effect Acetylation of histones Activator can be also a repressor

Repressors Repressors can operate in different ways

Repressors and activators attract histone acetylases and deacetylases

Control of regulatory proteins Some ways in which the activity of gene regulatory proteins is regulated in eucaryotic cells

Posttranscriptional modifications RNA capping and RNA polyadenylation increase the stability of mRNA Alternative splicing RNA editing

Alternative splicing the primary transcript can be spliced in various ways, removing not only all introns but also certain exons, to produce different mRNAs, depending on the cell type in which the gene is being expressed, or the stage of development of the organism –enables eucaryotes to increase the coding potential of their genomes –creates plasticity of eucaryotic genetic information –influence of environmental protein factors on RNA splicing –allows different proteins to be produced from the same gene (1 gene – 5 proteins) –alternative splicing is tissue specific

RNA editing RNA editing - insertion or deletion of nucleotides or substitution of nucleotides in transcribed RNA –certain change of transcribed genetic information –it can result in the appearance of new initiation and stop codons –gRNA (guide RNA)

RNA editing

Translation Each mRNA molecule is eventually degraded (RNA degradation) within the cell The lifetime of corresponding mRNA affects the expression of particular gene (longer lifetime of the mRNA means higher level of translated protein and vice versa) The lifetime of mRNA is regulated by nucleotide sequences in the 3' untranslated region of mRNA Translation can be also regulated by specific protein binding to mRNA –eg. IRP (iron regulatory protein)/IRE (iron responsive element) system

Regulation on the principle of stabilization of mRNA Iron-dependent regulation of the stability of transferrin-receptor mRNA system IRP/IRE

Regulation on the principle of inhibition of translation Iron-dependent regulation of translation of ferritin mRNA system IRP/IRE

Posttranslational modifications Newly synthesized polypeptide chain can be modified in several ways including both the cleavage of polypeptide chain and the binding of molecules –Removal of methionine from the N end: every newly synthesized polypeptide chain starts with methionine –Removal of single sequence: the signal sequence is a sequence of aminoacids serving as a signal for transfer to required location –Proteolytic cleavage: the formation of functional protein by cleavage of a precursor polypeptide chain (proinsulin → insulin)

–Formation of disulfide bonds: they are formed between adjacent cysteines. They help to stabilize protein structure –Chemical modification of amino acids: phosphorylation (binding of phosphate) hydroxylation (binding of –OH group) –Glycosylation: the binding of oligosacharide chains (glycoproteins) –Binding of prosthetic groups: the binding of prosthetic group (nonamino acid/nonprotein molecule) can be required for the functioning of protein (heme in hemoglobin)

Insulin

Protein degradation - proteasome Protein degradation – regulation of the amount of particular protein within the cell Individual proteins vary in their life span Most proteins in cells are degraded by proteasomes - Proteasome is a large complex of proteolytic enzymes forming kind of cylinder - Proteins are marked for degradation by the covalent binding of a small protein ubiquitin