{ Gene Expression Regulation Abira Khan

 Regulation of What, Where, When & How much is expressed  The cellular concentration of a protein depends on: 1. Synthesis of the primary RNA transcript (transcription) 2. Posttranscriptional modification of mRNA 3. mRNA degradation 4. Proteins synthesis (translation) 5. Posttranslational modification of proteins 6. Protein targeting and transport 7. Protein degradation  Control at the level of transcription initiation will be a major focus Why & Where Regulation?

 Housekeeping genes: Constitutive gene expression  Rest of the genes: Regulated gene expression  Regulation of 2 types: 1. Inducible genes: Induction 2. Repressible genes: Repression  RNA polymerase binds to DNA at promoter: For housekeeping genes, the RNA polymerase-promoter interaction strongly influences the rate of transcription initiation. Expression of other genes further modulated by regulatory proteins  Transcription initiation is regulated by proteins binding to/near promoter: 3 types- Specificity factors (RNA Pol), Repressors (Operators), Activators (Enhancers)  Regulatory proteins have DNA binding domains: Helix-turn-helix, Zinc finger, Homeodomain  Regulatory proteins have protein binding domains: Leucine zipper, Basic helix-loop-helix  Cis-acting elements at a gene are DNA sequences that serve as attachment sites for the DNA-binding proteins that regulate the initiation of transcription  Trans-acting elements are genes located somewhere other than at the target gene and encode proteins (or, in rare cases, RNAs) that interact directly or indirectly with the target gene’s cis-acting elements to activate or repress expression of the target gene Principles of Regulation

 Bacterial genes are clustered and regulated in operons  Regulatory mechanisms of 2 general types: 1. Rapid turn-on/turn-off in response to environmental change 2. Preprogrammed circuits/cascades of gene expression  Genes encoding enzymes of catabolic pathways often are expressed only in the presence of the substrates of the enzymes; their expression is inducible  Genes encoding enzymes of anabolic pathways usually are turned off in the presence of the end product of the pathway; their expression is repressible Regulation in Prokaryotes

 In positive control mechanisms, the product of the regulator gene is required to turn on the expression of one or more structural genes (genes specifying the amino acid sequences of enzymes or structural proteins)  In negative control mechanisms, the product of the regulator gene is necessary to shut off the expression of structural genes  The product of the regulator gene acts by binding to a site called the regulator protein-binding site (RPBS) adjacent to the promoter of the structural gene(s)  Whether or not a regulator protein can bind to the RPBS depends on the presence or absence of effector molecules in the cell: The effector molecules involved in induction of gene expression are called inducers; those involved in repression of gene expression are called co-repressors  The effector molecules (inducers and co-repressors) bind to regulator gene products (activators and repressors) and cause changes in the three-dimensional structures of these proteins: Allosteric transitions Positive & Negative Control

 Genes with related functions often are present in coordinately regulated genetic units called operons  The operon model, a negative control mechanism, was developed in 1961 by François Jacob and Jacques Monod  In the case of an inducible operon, the free repressor binds to the operator, turning off transcription  In the case of a repressible operon, the situation is reversed. The free repressor cannot bind to the operator. Only the repressor/effector molecule (co-repressor) complex is active in binding to the operator  Network of operons with a common regulator- Regulon Operon Model

Lactose Operon in E. coli

 Catabolite repression/Glucose effect: CAP+cAMP. The lac promoter contains two separate binding sites, one for RNA polymerase and one for the CAP/cAMP complex. in the absence of cAMP, CAP does not bind  cAMP acts as the effector molecule  High concentrations of glucose cause sharp decreases in the intracellular concentration of cAMP. Glucose prevents the activation of adenylcyclase, the enzyme that catalyzes the formation of cAMP from ATP  In the presence of glucose, lac operon transcription never exceeds 2% of the induced rate observed in the absence of glucose  By similar mechanisms, CAP and cAMP keep the arabinose (ara) and galactose ( gal ) operons of E. coli from being induced in the presence of glucose Lactose Operon in E. coli

1. Binding of RNA polymerase to its binding site in the lac promoter 2. Binding of CAP/cAMP to its binding site in the lac promoter 3. Binding of the lac repressor to the lac operators  The bending of the DNA by CAP/ cAMP promotes a more open site for RNA polymerase and thus enhanced binding and transcription of the structural genes.  Each tetrameric repressor binds two operator sequences simultaneously. One of the dimers binds to O1, and the other binds to either O2 or O3. In so doing, the repressor bends the DNA forming either a hairpin (O1 and O2) or a loop (O1 and O3) Protein-DNA Interactions at Lac Operon

Lac Operon at a Glance

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 Negative repressible operon  The expression of the trp operon is regulated at two levels: repression, which controls the initiation of transcription, and attenuation, which governs the frequency of premature transcript termination  Repression: In the absence of tryptophan (the co-repressor), RNA polymerase binds to the promoter region and transcribes the structural genes of the operon. In the presence of tryptophan, the co-repressor/repressor complex binds to the operator region and prevents RNA polymerase from initiating transcription of the genes in the operon  The rate of transcription of the trp operon in the derepressed state (absence of tryptophan) is 70 times the rate that occurs in the repressed state (presence of tryptophan) Tryptophan Operon in E. coli

 Attenuation: Occurs by control of the termination of transcription at a site near the end of the mRNA leader sequence. Occurs only in the presence of tryptophan-charged tRNATrp  The four leader regions that can base-pair to form these structures are: (1) nucleotides 60–68, (2) nucleotides 75–83, (3) nucleotides 110–121, and (4) nucleotides 126–134  2 possible secondary structures for the trp leader sequence: (1) region 1 paired with region 2 and region 3 paired with region 4 or (2) region 2 paired with region 3, leaving regions 1 and 4 unpaired  The pairing of regions 3 and 4 produces the transcription-termination hairpin  The 2 Trp codons in trpL are positioned such that in low concentrations of tryptophan, the ribosome will stall before it encounters the base-paired structure formed by leader regions 2 and 3  In the presence of sufficient tryptophan, transcription frequently terminates at the attenuator Tryptophan Operon in E. coli

 Either the frequency of initiation of translation or the rate of polypeptide chain elongation  Gene products may be produced in different amounts from the same transcript by several mechanisms 1. Unequal efficiencies of translational initiation are known to occur at the ATG start codons of different genes 2. Altered efficiencies of ribosome movement through intergenic regions of a transcript are quite common. Decreased translation rates often result from hairpins or other forms of secondary structure that impede ribosome migration along the mRNA molecule 3. Differential rates of degradation of specific regions of mRNA molecules also occur Translational Control

 Feedback inhibition: When the product of a biosynthetic pathway inhibits the activity of the first enzyme in the pathway, rapidly shutting off the synthesis of the product  In tryptophan biosynthetic pathway in E. coli, tryptophan is bound by the first enzyme in the pathway, anthranilate synthetase and completely arrests its activity  Allosteric transitions: Glutamine synthetase of E. coli has been shown to respond, either by activation or inhibition, to 16 different metabolites Posttranslational Regulatory Mechanisms

 Sigma (σ) subunit is required for the bacterial RNA polymerase to recognize a promoter. Most bacteria produce a whole range of sigma subunits, each of which can interact with the RNA polymerase core and direct it to a different set of promoters  This scheme permits one large set of genes to be turned off and a new set to be turned on simply by replacing one sigma subunit with another  Bacterial viruses often use it subversively to take over the host polymerase and activate several sets of viral genes rapidly and sequentially RNA Pol Subunits

 An increase in the transcription of the rpoH gene  An increase in the translation of σ32 mRNA stemming from greater stability of the rpoH mRNA  An increase in the stability and activity of the σ32 protein. Chaperones DnaJ/K bind to and inhibit σ32 under normal physiological conditions. When the temperature rises, these proteins bind to the large number of cellular proteins that become denatured, leaving σ32 free to associate with RNA polymerase  The inactivity of σ70 at high temperatures. Because of this inactivity, σ70 does not compete with σ 32 in forming the RNA polymerase holoenzyme  At high temperatures, another sigma factor, σ24, recognizes a different promoter sequence at rpoH and transcribes the rpoH gene from that promoter. Although σ24 is always present in the cell, its own transcription (mediated by the σ24 holoenzyme) increases with heat shock and the appearance of denatured proteins Heat Shock Response in E. coli

 Transcription of genes by RNA polymerases containing σ54 is regulated solely by activators binding to enhancers  In response to low levels of glutamine, NtrB phosphorylates dimeric NtrC, which then binds to an enhancer upstream of the glnA promoter  Enhancer-bound phosphorylated NtrC then stimulates the σ54-polymerase bound at the promoter to separate the DNA strands and initiate transcription  NtrC has ATPase activity  E. coli proteins PhoR and PhoB, which regulate transcription in response to the concentration of free phosphate Bacterial Responses Controlled by 2 Component Regulatory Systems

 Extensive DNA damage in bacteria triggers SOS response, produces DNA damage repair proteins  Regulatory proteins- RecA, LexA  LexA inhibits transcription of all SOS genes  Heavy DNA damage leads to ssDNA, RecA binds these and acts as coprotease for the autocleavage of LexA at Ala-Gly bond  Also leads to cleavage of repressors which maintain virus DNA in lysogenic state- so lytic cycle of virus leads to production of new virus particles Destruction of Repressor Proteins

 rProtein binds to rRNA, excessive rProtein bonds to own mRNA, acts as translational repressor  In E. coli, rRNA synthesis from 7 operons responds to cellular growth rate and to changes in the availability of crucial nutrients, particularly amino acids: Stringent response  Amino acid starvation leads to the binding of uncharged tRNAs to the ribosomal A site; this triggers a sequence of events that begins with the binding of an enzyme called stringent factor (RelA protein) to the ribosome. It catalyzes formation of the unusual nucleotide guanosine tetraphosphate, which binds to RNA Pol and reduces rRNA synthesis Ribosomal Protein Synthesis

 Act in cis/trans  Sigma s in E. coli, hairpin upstream of coding region inhibits ribosome binding  Under certain stress conditions, one or both of two small special-function RNAs, DsrA (downstream region,A) and RprA (Rpos regulator RNA A), are induced, bind to hairpin and relieves repression  Small RNA, OxyS (oxidative stress gene S), is induced under conditions of oxidative stress and inhibits the translation of rpoS  Hfq, an RNA chaperone facilitates RNA-RNA pairing  Riboswitches act in cis, bind a ligand RNA s & Riboswitches

 Salmonella typhimurium switch between two distinct flagellin proteins (FljB and FliC) roughly once/1,000 generations: Phase variation  Periodic inversion of a segment of DNA containing the promoter for a flagellin gene. The 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  When the DNA segment is in one orientation, the gene for FljB flagellin and the gene encoding a repressor (FljA) are expressed. the repressor shuts down expression of the gene for FliC flagellin  When the DNA segment is inverted, the fljA and fljB genes are no longer transcribed, and the fljC gene is induced as the repressor becomes depleted  The Hin recombinase, encoded by the hin gene in the DNA segment that undergoes inversion is expressed when the DNA segment is in either orientation Genetic Recombination

 Principles of Biochemistry- Lehninger- 5th Edition, Chapter 28  Principles of Genetics- Peter Snustad- 6th edition, Chapter 18  Genetics, from Genes to Genomes - Hartwell- 2nd edition, Chapter 16  Molecular Cell Biology- Lodish- 5th Edition, Chapter 4  Molecular Biology of the Cell- Alberts- 5th edition, Chapter 7 References