Gene Regulation (1)

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Gene Regulation:

Gene Regulation © Biotecnika Info Labs Pvt. Ltd.

Principles of Gene Regulation: Some Common terms:

Principles of Gene Regulation: Some Common terms Housekeeping genes expressed at a more or less constant level in virtually every cell of a species or organism Constitutive gene expression Unvarying expression of a gene Regulated gene expression Gene expression in response to molecular signals Inducible Gene products Gene products that increase in concentration under particular molecular circumstances Induction process of increasing the expression of Gene Repressible Gene Product Gene products that decrease in concentration in response to a molecular signal Repression process of decreasing the expression of Gene

Gene expression is controlled by regulatory proteins (Regulators) - are DNA binding protein - Types: :

Gene expression is controlled by regulatory proteins (Regulators) - are DNA binding protein - Types : Positive regulators (Activators) Binds to activator binding site Helps to bring RNA Polymerase to the promoter (called as recruitment) Negative regulators (Repressors ) Bind to operators Interacts with RNA Polymerase with allostery

Positive regulators (Activators) Activators work by - Interacting with Polymerase or - by allostery:

Positive regulators (Activators) Activators work by - Interacting with Polymerase or - by allostery Interaction with Polymerase Has two surfaces for interaction One with DNA Other with RNA Polymerase Binds to activator binding site Helps to bring RNA Polymerase to the promoter (called as recruitment) Binding is Cooperative E.g. σ factor in E.coli Allostery Causes confirmational changes Binding of activator causes transition from closed complex to open complex E.g. Interaction of NtrC (at gln promoter) with RNA Polymerase E.g. Interaction of MerR (at merT promoter) with promoter DNA

DNA binding domains of regulatory proteins:

DNA binding domains of regulatory proteins Helix- turn- helix Comprises about 20 amino acids in two short -helical segments each seven to nine amino acid residues long, separated by a β turn

Zinc finger :

Zinc finger about 30 amino acid residues form an elongated loop held together at the base by a single Zn2+ ion , which is coordinated to four of the residues four Cys, or two Cys and two His. The zinc does not itself interact with DNA; the coordination of zinc with the amino acid residues stabilizes motif.

Homeodomain :

Homeodomain 60 amino acids long Called so because discovered in homeotic genes genes that regulate the development of body patterns highly conserved DNA-binding segment of the domain is related to the helix-turn-helix motif Found as one of the α helices (red), stacked on two others, Ist α helix can be seen protruding into the major groove Note: DNA sequence that encodes homeodomain is known as the homeobox

Protein-Protein Interaction Domains of regulatory proteins:

Protein-Protein Interaction Domains of regulatory proteins Leucine zipper motif is an amphipathic helix with a series of hydrophobic amino acid residues concentrated on one side with the hydrophobic surface forming the area of contact between the two polypeptides of a dimer . occurrence of Leu residues at every seventh position , forming a straight line along the hydrophobic surface.

Basic helix-loop-helix:

Basic helix-loop-helix Has conserved region of about 50 amino acid residues important in both DNA binding and protein dimerization can form two short amphipathic α helices linked by a loop of variable length, the helix-loop- helix helix-loop-helix motifs of two polypeptides interact to form dimers

Types of regulation -Negative regulation - Positive regulation:

Types of regulation -Negative regulation - Positive regulation Negative regulation Regulation by means of a repressor protein that blocks transcription Repressor binding to DNA is regulated by a molecular signal Effector small molecule or a protein binds to the repressor causes a conformational change Positive regulation Regulation by means of a activator protein which enhances the activity of RNA polymerase at a promote r Activator either binds to Activator binding sites (near to promoter)or to enhancer (distant from the promoter)

Gene Regulation in Prokaryotes:

Gene Regulation in Prokaryotes Regulation at transcription initiation

Lac operon:

Lac operon

Regulatory proteins of Lac operon: Both are DNA binding proteins:

Regulatory proteins of Lac operon: Both are DNA binding proteins Lac Repressor Encoded by Lac I Under constitutive expression Has its own promoter for expression Mediates lactose signal Bind operator & repress transcription in absence of lactose Binds to 21 bp long sequence (in promoter) in twofold symmetry Binds as tetramer Only 2 monomer interacts with DNA at a time

CAP (activator) :

CAP (activator) Catabolite activator protein Also known as CRP (cAMP Receptor Protein) Gene located at other site on bacterial genome Mediates effect of Glucose Activate the genes in absence of Glucose Binds to 60bp upstream of start site Binds as a dimer Binding is cooperative Note: CAP can work together at different genes viz. Lac & gal (Combinatorial Control)

Interaction of Lac regulators with DNA:

Interaction of Lac regulators with DNA Interacts with helix turn helix motif First helix is recognition helix Fits into major groove of DNA Interaction involves Direct H-Bond Indirect H bonds Vanderwaals forces Second helix Sits across major groove Makes contact with DNA backbone

Activities of Lac regulators are controlled by their signals:

Activities of Lac regulators are controlled by their signals Signal for Lac repressor : Allolactose formed from Lactose by the activity of β - galactosidase Inhibits Lac repressor Binds to Lac repressor at distinct site Signal for CAP: cAMP Produced from ATP

Positive Regulation of Lac Operon:

Positive Regulation of Lac Operon

Process of Regulation of Lac operon:

Process of Regulation of Lac operon

Regulation at steps after transcription initiation:

Regulation at steps after transcription initiation trp operon

Binding of Trp repressor to Operator of Trp:

Binding of Trp repressor to Operator of Trp

Regulation of Trp Operon - in the presence of Trp:

Regulation of Trp Operon - in the presence of Trp

Transcriptional attenuation in the trp operon:

Transcriptional attenuation in the trp operon

When tryptophan levels are high, the ribosome quickly translates sequence 1 (open reading frame encoding leader peptide) and blocks sequence 2 before sequence 3 is transcribed. Continued transcription leads to attenuation at the terminator-like attenuator structure formed by sequences 3 and 4.:

When tryptophan levels are high , the ribosome quickly translates sequence 1 (open reading frame encoding leader peptide) and blocks sequence 2 before sequence 3 is transcribed. Continued transcription leads to attenuation at the terminator-like attenuator structure formed by sequences 3 and 4.

When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence 1. Formation of the paired structure between sequences 2 and 3 prevents attenuation, because sequence 3 is no longer available to form the attenuator structure with sequence 4. The 2:3 structure, unlike the 3:4 attenuator, does not prevent transcription:

When tryptophan levels are low , the ribosome pauses at the Trp codons in sequence 1 . Formation of the paired structure between sequences 2 and 3 prevents attenuation, because sequence 3 is no longer available to form the attenuator structure with sequence 4. The 2:3 structure , unlike the 3:4 attenuator, does not prevent transcription

SOS response:

SOS response Extensive DNA damage in the bacterial chromosome triggers the induction of many distantly located genes key regulatory proteins are the RecA protein and the LexA repressor inhibits transcription of all the SOS genes

Slide32:

SOS response in E. coli 1 When DNA is extensively damaged (e.g., by UV light), DNA replication is halted and the number of single-strand gaps in the DNA increases . 2 RecA protein binds to this damaged, single-stranded DNA, activating the protein’s coprotease activity. 3 While bound to DNA, the RecA protein facilitates cleavage and inactivation of the LexA repressor. When the repressor is inactivated, the SOS genes, including recA, are induced; RecA levels increase 50- to 100-fold. Autocatalytic nature of LexA LexA catalyzes its own cleavage at a specific Ala–Gly peptide bond, producing two roughly equal protein fragments. Action of RecA RecA is not a protease in the classical sense, but its interaction with LexA facilitates the repressor’s self-cleavage reaction. This function of RecA is sometimes called a coprotease activity

Genes Induced as Part of the SOS Response in E. coli:

Genes Induced as Part of the SOS Response in E. coli

Translational Level control:

Translational Level control Ribosomal Protein Operons

Why Regulation of ribosome synthesis needed by bacteria?:

Why Regulation of ribosome synthesis needed by bacteria? Regulation of protein synthesis is not done by altering ribosome an increased cellular demand for protein synthesis is met by increasing the number of ribosomes r-genes 52 genes encode the r-proteins occur in at least 20 operons, each with 1 to 11 genes. Some of these operons also contain the genes for the subunits of DNA primase, RNA polymerase and protein synthesis elongation factors r-protein operons are regulated primarily through a translational feedback mechanism

Translational feedback in some ribosomal protein operons:

Translational feedback in some ribosomal protein operons

Regulation of genes by Genetic Recombination:

Regulation of genes by Genetic Recombination Regulation of flagellin genes in Salmonella : phase variation

Salmonella typhimurium:

Salmonella typhimurium inhabits the mammalian intestine moves by rotating the flagella on its cell surface copies of the protein flagellin ( M r 53,000) that make up the flagella are prominent targets of mammalian immune systems evades the immune response by phase variation they switch between two distinct flagellin proteins ( FljB and FliC ) roughly once every 1,000 generations switch is accomplished by periodic inversion of a segment of DNA containing the promoter for a flagellin gene inversion is a site-specific recombination reaction mediated by the Hin recombinase at specific 14 bp sequences ( hix sequences) at either end of the DNA segment

Process of Regulation of flagellin genes in Salmonella:

Process of Regulation of flagellin genes in Salmonella

Regulation of Gene Expression in Eukaryotes:

Regulation of Gene Expression in Eukaryotes

Eukaryotic Gene regulation different from prokaryotes???:

The transcriptional ground state is restrictive strong promoters are generally inactive in vivo in the absence of regulatory proteins Access to eukaryotic promoters is restricted by the structure of chromatin Eukaryotic cells have larger, more complex multimeric regulatory proteins than do bacteria. Transcription in the eukaryotic nucleus is separated from translation in the cytoplasm in both space and time Eukaryotic Gene regulation different from prokaryotes???

Transcriptionally Active Chromatin Is Structurally Distinct from Inactive Chromatin:

Transcriptionally Active Chromatin Is Structurally Distinct from Inactive Chromatin Nucleosomes are entirely absent in some regions that are very active in transcription, such as the rRNA genes Histones within transcriptionally active chromatin and heterochromatin also differ in their patterns of covalent modification . The core histones of nucleosome particles (H2A, H2B, H3, H4) are modified by irreversible methylation of Lys residues , phosphorylation of Ser or Thr residues, acetylation, or attachment of ubiquitin DNA in transcriptionally active chromatin tends to be under methylated . CpG sites under methylated in cells from tissues where the genes are expressed than in those where the genes are not expressed

Chromatin remodeling- transcription-associated structural changes in chromatin :

Chromatin remodeling- transcription-associated structural changes in chromatin done by enzymes covalently modify the core histones of the nucleosome and remodel nucleosomes on the DNA use the chemical energy of ATP

Enzymes involved in chromatin remodelling:

Enzymes involved in chromatin remodelling Histone acetyltransferases acetylates Lys residue of Histone core Reduces affinity of the entire nucleosome for DNA SWI/SNF move or displace nucleosomes , hydrolyzing ATP contains 11 polypeptides create hypersensitive sites in the chromatin stimulate the binding of transcription factors Not required for every gene NURF ATP-dependent enzyme complex Action is same as that of SWI/SNF

Molecules involved in regulation of gene expression in eukaryotes:

Molecules involved in regulation of gene expression in eukaryotes Basal transcription factors , required at every Pol II promoter e.g. TFIID, TFIIB etc DNA binding transactivators, binds to enhancers or UASs (upstream activator sequence) facilitate transcription Coactivators act indirectly Do not bind to the DNA are required for essential communication between the DNA-binding transactivators and the complex composed of Pol II and the general transcription factors.

Interaction between molecules involved in Transcription Initiation in Eukaryotes:

Interaction between molecules involved in Transcription Initiation in Eukaryotes

Gal Operon in Yeast:

Gal Operon in Yeast

Galactose metabolism in yeast:

Galactose metabolism in yeast

DNA Binding transactivators in Eukaryotes :

DNA Binding transactivators in Eukaryotes Gal4p contains a zinc fingerlike structure in its DNA-binding domain, near the amino terminus; this domain has six Cys residues that coordinate two Zn2+. The protein functions as a homodimer and binds to UAS G , (a palindromic DNA sequence about 17 bp long). has a separate activation domain with many acidic amino acid residues.

Contd… :

Contd… Sp1 M r 80,000 DNA-binding transactivator for a large number of genes in higher eukaryotes. DNA binding site, the GC box (consensus sequence GGGCGG), is usually quite near the TATA box. DNA-binding domain of the Sp1 protein is near its carboxyl terminus and contains three zinc fingers. Two other domains in Sp1 function in activation, and are notable in that 25% of their amino acid residues are Gln.

Contd…:

Contd… CTF1 bind a sequence called the CCAAT site (its consensus sequence is TGGN 6 GCCAA). The DNA-binding domain of CTF1 contains many basic amino acid residues, the binding region is arranged as an α helix. This protein has neither a helix turn- helix nor a zinc finger motif; its DNA-binding mechanism is not yet clear. has a proline-rich activation domain, with Pro accounting for more than 20% of the amino acid residues.

Regulation of gene expression by Intercellular and Intracellular Signals in eukaryotes:

Regulation of gene expression by Intercellular and Intracellular Signals in eukaryotes General mechanism by which steroid and thyroid hormones, retinoids, & vitamin D regulate gene expression.

Slide56:

Hormone-receptor complex binds to the DNA as a dimer, with the zinc finger domains of each monomer recognizing one of the six-nucleotide sequences Ligand-binding region of the receptor protein — always at the carboxyl terminus—is quite specific to the particular receptor

Eukaryotic gene regulation By Phosphorylation of Nuclear Transcription Factors :

Eukaryotic gene regulation By Phosphorylation of Nuclear Transcription Factors

Slide59:

Translational Repression in eukaryotes

Three main mechanisms of translational repression in Eukaryotes:

Three main mechanisms of translational repression in Eukaryotes Phosphorylation of Initiation factors by protein kinases inactivates them Action of translational repressors , bind directly to mRNA in the 3 untranslated region (3’UTR). Interact with other translation initiation factors bound to the mRNA or with the 40S ribosomal subunit to prevent translation initiation Disruption of the interaction between eIF4E and eIF4G by binding proteins

Slide61:

A model for transcriptional repression

Posttranscriptional Gene Silencing:

Posttranscriptional Gene Silencing Mediated by RNA Interference

Slide63:

Part of RNA

Gene regulation during development:

Gene regulation during development Drosophila: A case study

Development Is Controlled by Cascades of Regulatory Proteins:

Development Is Controlled by Cascades of Regulatory Proteins Development requires transitions in morphology and protein composition that depend on tightly coordinated changes in expression of the genome. More genes are expressed during early development than in any other part of the life cycle

Slide67:

life cycle of the fruit fly includes complete metamorphosis during its progression from an embryo to an adult the most important characteristics of the embryo are its polarity the anterior and posterior parts of the animal are readily distinguished, as are its dorsal and ventral parts metamerism the embryo body is made up of serially repeating segments, each with characteristic features During development, these segments become organized into a head, thorax, and abdomen.

Slide68:

After fertilization egg nucleus divides the nuclear descendants continue to divide in synchrony every 6 to 10 min. Plasma membranes are not formed around the nuclei, which are distributed within the egg cytoplasm (or syncytium) Before fertilization mRNAs and proteins originating in the nurse and follicle cells are deposited in the egg cell,

Early development in Drosophila:

Early development in Drosophila

Early development in Drosophila:

Early development in Drosophila Between the 8th and 11th rounds of nuclear division, syncytial blastoderm is formed the nuclei migrate to the outer layer of the egg, forming a monolayer of nuclei surrounding the common yolk-rich cytoplasm After a few additional divisions, Blastoderm is formed membrane invaginations surround the nuclei to create a layer of cells that form the cellular. At this stage, the mitotic cycles in the various cells lose their synchrony. The developmental fate of the cells is determined by the mRNAs and proteins originally deposited in the egg by the nurse and follicle cells.

Early development in Drosophila:

Early development in Drosophila Morphogens - Proteins that, through changes in local concentration or activity, cause the surrounding tissue to take up a particular shape or structure are the products of pattern regulating genes 3 pattern regulating genes function in successive stages of development to specify the basic features of the Drosophila embryo’s body Maternal genes Segmentation genes, and homeotic genes

Maternal genes:

Maternal genes expressed in the unfertilized egg within the nurse and follicle cells, and some in the egg itself The resulting maternal mRNAs remain dormant until fertilization. These provide most of the proteins needed in very early development, until the cellular blastoderm is formed. Also direct the spatial organization of the developing embryo at early stages, establishing its polarity. establish two axes— anterior-posterior & dorsal-ventral define which regions of the radially symmetric egg will develop into the head and abdomen and the top and bottom of the adult fly. E.g. bicoid gene & nanos gene

The anterior-posterior axis in Drosophila is defined by the products of the bicoid gene & nanos gene:

The anterior-posterior axis in Drosophila is defined by the products of the bicoid gene & nanos gene bicoid genes- gene product is a major anterior morphogen mRNA from the bicoid gene is synthesized by nurse cells and deposited in the unfertilized egg near its anterior pole this mRNA is translated soon after fertilization, and the Bicoid protein diffuses through the cell to create, by the seventh nuclear division, a concentration gradient radiating out from the anterior pole Acts as transcriptional activator activates the expression of a number of segmentation genes Acts as translational repressor Inactivates certain mRNAs

Slide74:

Nanos genes. gene product is a major posterior morphogen . its mRNA is deposited at the posterior end of the egg Nanos protein is a translational repressor Other gene products Pumilio, Hunchback, and Caudal proteins Regulatory circuits of the anterior-posterior axis in a Drosophila egg.

Segmentation genes:

Segmentation genes transcribed after fertilization, direct the formation of the proper number of body segments. At least three subclasses of segmentation genes act at successive stages: gap genes divide the developing embryo into several broad regions regulated by the products of one or more maternal genes encode transcription factors that affect the expression of other segmentation or (later) homeotic genes pair-rule genes together with segment polarity genes define 14 stripes that become the 14 segments of a normal embryo

Homeotic genes:

Homeotic genes expressed after Segmentation genes are expressed They specify which organs & appendages will develop in particular body segments. Note: embryogenesis takes about a day to complete, but pattern regulating genes are activated during the first four hours.

Gene Regulation in Bacteriophages:

Gene Regulation in Bacteriophages Gene Regulation in T 4 Bacteriophage & Gene Regulation in λ Bacteriophage

Gene Regulation in T4 Bacteriophage:

Gene Regulation in T 4 Bacteriophage

Slide79:

After phage infection in E.coli cells the synthesis of host DNA, RNA, and protein is halted, the cell is forced to make viral constituents E. coli RNA polymerase starts synthesizing phage mRNA within 2 minutes which direct the synthesis of the protein factors and enzymes required to take over the host cell and manufacture viral nucleic acids. Virus enzyme catalyzes the transfer of an ADP-ribosyl group from NAD to an α -subunit of RNA polymerase . inhibits the transcription of host genes and promotes virus gene expression.

Slide80:

Early virus specific enzymes degrade host DNA to nucleotides, halts host gene expression and provides raw material for virus DNA synthesis Regulation of Gene expression in T4 phage Inhibition of Late genes ADP Ribosylation of second α -subunit of RNA Polymerase the second α -subunit receives an ADP-ribosyl group this turns off some of the early T4 genes Activation of Late Genes Done by motA protein product of one early gene, stimulates transcription of late genes, one of which produces the sigma factor gp55. Helps RNA polymerase bind to late promoters and transcribe late genes

Gene Regulation in λ Bacteriophage:

Gene Regulation in λ Bacteriophage

Lytic & Lysogenic cycle:

Lytic & Lysogenic cycle

Slide84:

Note: P R & P L are strong promoters while P RM is weak promoter P RE present after cII gene Promoter for cI gene

Slide85:

Relative sites of promoter & operator P R - R ightward P romoter; P L - L eft ward P romoter P RM - P romoter for R epressor M aintenance

Lambda repressor:

Lambda repressor coded for by the cI gene Under constitutive expression in a lysogen Structure 236 amino acids long and folds into a dumbbell shape with globular domains at each end One domain binds to DNA, the other domain binds to another repressor molecule to generate a dimer Binds to DNA as a dimer binds to the operators O L and O R , thereby blocking RNA polymerase activity

Autoregulation of repressor:

Autoregulation of repressor Positive Autoregulation Activates its own expression by binding to P RM as P RM is weak promoter Negative Autoregulation Represses its own expression by blocking P RM (binds to O R3 ) Seen at maximum Repressor concentration

Regulatory proteins & their binding sites:

Regulatory proteins & their binding sites Cro protein Has more affinity for O R3 Has same affinity for O R2 & O R1 Binds as a dimer Represses formation of Repressor as its binding overlaps P RM which is promoter for Lambda Repressor Directs viral genome towards lytic cycle Lambda Repressor Has more affinity for O L1 & O R1 Has same affinity for O R2 & O R3 Binds as dimer & Binding is Co-operative Directs virus towards lysogeny Note: Repressor udergoes autocleavage to get inactivated when adverse condition arises (during SOS Response- when the bacterial cell is under life threatening situation)

Sequence of events during Phage Infection :

Sequence of events during Phage Infection Phage genome entry Phage genome circularization Transcription commences, the cII and cIII proteins accumulate cII stimulates RNA polymerase binding at upstream of P R (cIII protects cII from degradation)

Slide90:

Cro protein accmulates cII activates Int gene (Integrase) cI gene expression starts cI repressor blocks O R1 & O L1 Transcription of other gene halts Competition between cro protein & cI Repressor to decide fate of phage

Choice of Lytic & Lysogeny:

Choice of Lytic & Lysogeny Major protein involved in the choice of Lytic & Lysogeny is cII protein Unstable protein Enhances expression of cI gene (Repressor) Detected & degraded by by a specific bacterial proteases FtsH

Slide94:

When conditions are optimum for bacterial growth Lytic cycle is favoured Case I cII proteins are degraded by FtsH protein Hence less quantity of repressor is produced Case II ( Antitermination ) Mediated by Protein N & protein Q Binds to RNA Polymerase (via mRNA which is under synthesis process) & makes it resistant to terminators Case III ( Retroregulation ) Degradation of Integrase mRNA by antitermination Antitermination produces longer ds stem of mRNa which invites nuclease action by nucleases of bacteria

Slide95:

When conditions are poor for bacterial growth Lysogeny is favoured Case I cII proteins are less degraded by FtsH protein Hence more quantity of repressor is produced Case II cII proteins increase concentration of Integrase which integrates the viral genome to the bacterial genome by site specific recombination Case III P AQ RNA acts as antisense to Q RNA & enhances its degradation

Combinatorial Control in Yeast:

Combinatorial Control in Yeast

Forms of yeast:-:

Forms of yeast:- 2 haploid forms- a & α 1 diploid form- formed after a & α fuses A cells encode regulatory protein a1 α cells encodes regulatory protein α 1 & α 2 Other regulatory protein is Mcm1

Control of cell specific genes in yeast:

Control of cell specific genes in yeast

Thanks:

Thanks Please visit Chapter-3 of http://study.biotecnika.org for more details

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