Lecture 14a: General Principles of Gene Expression Regulation in E. Coli PDF

Lecture 14a General principles of regulation of gene expression Regulation of gene expression in E. Coli Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 28: p. 1054-1075 REGULATION OF GENE EXPRESSION Key topics: – General principles – Regulation of...

Lecture 14a General principles of regulation of gene expression Regulation of gene expression in E. Coli Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 28: p. 1054-1075 REGULATION OF GENE EXPRESSION Key topics: – General principles – Regulation of gene expression in bacteria DNA elements that control transcription Protein factors that control transcription Lac operon as a model for regulation Protein motifs/domains involved in regulation of gene expression GENERAL PRINCIPLES OF REGULATION OF GENE EXPRESSION HOW TO CONTROL PROTEIN’S ACTIVITY IN THE CELL How much primary RNA transcript to make How to process this RNA into mRNA How rapidly to degrade the mRNA How much protein to make from this mRNA How efficiently to target the protein to its location How to alter the intrinsic activity of this protein How rapidly to degrade the protein PRINCIPLES OF GENE REGULATION (1) The cellular concentration of a protein is determined by a delicate balance of at least 7 processes, each having several points of regulation: 1. Synthesis of the primary RNA transcript (transcription). 2. Postranscriptional modification of mRNA 3. mRNA degradation 4. Protein synthesis (translation) 5. Postranslational modification of proteins 6. Protein targeting and transport 7. Protein degradation Gene expression can be: - Constitutive (unvarying) - Regulated (varying) - relies on precise protein-DNA and protein-protein contacts - Inducible (induction) - Repressible (repression) Processes affecting the steady-state concentration of a protein (1 to 7) PRINCIPLES OF GENE REGULATION (2) Housekeeping gene – Under constitutive expression – Constantly expressed in almost all cells Regulated gene – Levels of the gene product rise and fall with the needs of the organism – Such genes are inducible able to be turned on – and repressible able to be turned off RNA POLYMERASE BINDING TO PROMOTERS IS A MAJOR TARGET OF REGULATION RNA polymerases bind to promoter sequences near starting point of transcription initiation The RNA Pol-promoter interaction greatly influences the rate of transcription initiation Regulatory proteins (transcription factors) work to enhance or inhibit this interaction between RNA pol and the promoter DNA SMALL-MOLECULE EFFECTORS CAN REGULATE ACTIVATORS AND REPRESSORS Repressors proteins reduce RNA Pol-promoter interactions or block the polymerase from binding to the promoter – Binding sites in DNA for repressors are called operators – In bacteria, operators are found usually near a promoter but further away in many eukaryotes Activators proteins improve contacts between RNA Pol and the promoter. – Binding sites in DNA for activators are called enhancers – In bacteria, enhancers are usually adjacent to promoter (-60 region) – Often adjacent to promoters that are “weak” (bind RNA Pol weakly) and so the activator is necessary – In eukaryotes, enhancers may be very distant from promoter Effectors (small molecules) can bind to repressor or activator and induce a conformational change – Change may increase or decrease repressor (or activator) affinity for the operator and thus may increase or decrease transcription NEGATIVE VS. POSITIVE REGULATION Negative regulation involves repressors (see lac operon) – Example: Repressor binds to DNA and shuts down transcription – Alternative: Signal (effector) causes repressor to dissociate from DNA; transcription induced (Despite opposite effects on transcription, both are negative regulation) Positive regulation involves activators (see lac operon) – Example: CRP (or CAP) binds to DNA and stimulates transcription in presence of a signal (effector), cAMP – Alternative: Absence of cAMP causes CRP to dissociate from DNA; transcription is inhibited (Despite opposite effects on transcription, both are positive regulation) NEGATIVE REGULATION Repressor binds to the operator in the absence of the molecular signal; the external signal causes dissociation of the repressor to permit transcription. Repressor binds in the presence of the signal; the repressor dissociates and transcription ensues when the signal is removed. POSITIVE REGULATION UP element Activator binds in the absence of the molecular signal and transcription proceeds; when the signal is added, the activator dissociates and transcription is inhibited Activator binds in the presence of the signal; it dissociates only when the signal is removed. PRINCIPLES OF GENE REGULATION: A RECAP At least 3 types of proteins regulate transcription initiation by RNA Polymerase: 1. Specificity factors (Ex: s subunit of E. coli RNA Polymerase) alter the specificity of RNA polymerase for a given promoter (Ex: Heat Shock Promoter). 2. Repressors (impede access of RNA Polymerase to the promoter) = Negative regulation 3. Activators (enhance the interaction of RNA-polymerase with the promoter) = Positive regulation - Repressors bind to operators binding sites (near or within the promoter) - Activators bind to activator binding sites (adjacent to the promoter) - Repressors and activators binding to DNA is regulated by a molecular signal (effector) - “positive” and “negative” regulation refer to the type of regulatory protein involved (not the effect on transcription): the bound protein either facilitates or inhibits transcription. In either case, addition of the molecular signal may increase or decrease transcription, depending on its effect on the regulatory protein. REGULATION OF GENE EXPRESSION IN E. COLI CONSENSUS SEQUENCE OF MANY E. COLI PROMOTERS RNA polymerase binds to DNA at promoters Most base substitutions in the –10 and –35 regions have a negative effect on promoter function. Some promoters also include the UP (Upstream Promoter) element, involved in binding transcription factors that modulate transcription By convention, DNA sequences are shown as they exist in the non-template strand, with the 5′ terminus on the left. Nucleotides are numbered from the transcription start site, with positive numbers to the right (in the direction of transcription) and negative numbers to the left. N indicates any nucleotide MECHANISMS TO REGULATE TRANSCRIPTION Use of specificity factors: σ factors – These factors recognize different classes of promoters – This allows coordinated expression of different sets of genes Binding other proteins (Transcription Factors, TFs) to promoters – TFs recognize promoters of specific genes – TFs may bind small signaling molecules – TFs may undergo post-translational modifications – TF’s affinity toward DNA is altered by ligand binding or posttranslational modifications Allows expression of specific genes in response to signals in the environment REGULATION BY SPECIFICITY FACTORS SUCH AS s SUBUNITS OF RNA POLYMERASE Specificity factors alter RNA polymerase’s affinity for certain promoters Example: s subunit of E. coli RNA Polymerase – Most E. coli promoters are recognized by s70 – This subunit can be replaced by one of six additional specificity factors Example: Heat shock will lead to replacement of s70 by s32 and direct RNA Pol to different promoters (heat shock promoters) EXAMPLE: SPECIFICITY OF s SUBUNIT IN HEAT SHOCK TO INDUCE TRANSCRIPTION OF NEW PRODUCTS TO PROTECT CELL Occurs when bacteria are subject to heat stress s32 replaces s70 in RNA Pol – Causes RNA Pol to bind to different set of promoters à transcription of new products including chaperones that keep proteins in correct conformation even during a heat stress Consensus sequence for promoters that regulate expression of the E. coli heat shock genes This system responds to temperature increases as well as some other environmental stresses, resulting in the induction of a set of proteins. Binding of RNA polymerase to heat shock promoters is mediated by a specialized σ subunit of the polymerase, σ32, which replaces σ70, in the RNA polymerase initiation complex. MANY BACTERIAL GENES ARE TRANSCRIBED AND REGULATED TOGETHER IN AN OPERON An operon is a cluster of genes sharing a promoter and regulatory sequences – Genes are transcribed together so mRNAs of bacteria may consist of several genes grouped in one mRNA (polycistronic mRNA) – Example: the lac operon REPRESENTATIVE BACTERIAL OPERON: GENES A, B, AND C ARE TRANSCRIBED ON ONE POLYCISTRONIC mRNA of 3 cistrons Typical regulatory sequences include binding sites for proteins that either activate or repress transcription from the promoter. THE LAC OPERON REVEALS MANY PRINCIPLES OF GENE REGULATION Shows how three genes for metabolism of lactose are regulated in a coordinated way: – b-galactosidase (lacZ) Cleaves lactose to yield glucose and galactose – Lactose permease (galactoside permease; lacY) Transports lactose into cell – Thiogalactoside transacetylase (lacA) CoA-dependent acetyltransferase specific for the 6hydroxyl group of certain pyranosides Function poorly understood Genes are regulated together as an operon Genes rely on negative regulation via a repressor LACTOSE METABOLISM IN E. COLI Uptake and metabolism of lactose require the activities of galactoside (lactose) permease and β-galactosidase. β-galactosidase also converts of lactose to allolactose (a lactose isomer) by transglycosylation (a minor reaction) ALLOLACTOSE IS AN INDUCER OF THE LAC OPERON Allolactose (an inducer) binds to the repressor, causing it to dissociate from operator – Made by b-galactosidase (isomerization of lactose into allolactose). – [Allolactose] ­ when [Lactose] ­ Allolactose REGULATION OF LACTOSE OPERON IN E. COLI When glucose is abundant and lactose is lacking, cells make only very low levels of enzymes for lactose metabolism – Transcription is repressed If glucose is scarce and cells are fed lactose, the cells can use it as their energy source – The cells suddenly express the genes for the enzymes for lactose metabolism – Transcription is no longer repressed (it is derepressed) INHIBITING THE TRANSCRIPTION OF THE LAC OPERON VIA A REPRESSOR PROTEIN sequestered Lac Promoter Lac Repressor bound Structure of the lac operon. a/ The lac operon. The lacI gene encodes the Lac repressor. The lac Z, Y, and A genes encode βgalactosidase, galactoside permease, and thiogalactoside transacetylase, respectively. P is the promoter for the lac genes, and PI is the promoter for the I gene. O1 is the main operator for the lac operon; O2 and O3 are secondary operator sites of lesser affinity for the Lac repressor. The inverted repeat to which the Lac repressor binds in O1 is shown in the inset. b/ The Lac repressor binds to the main operator and O2 or O3, apparently forming a loop in the DNA. A gene called lacI encodes a repressor called the Lac repressor – Has its own promoter PI So transcription of the repressor is independent of transcription of the enzymes the repressor regulates. – Repressor can bind to three operator sites (O1–O3) Lac repressor binds primarily to operator O1 – O1 is adjacent to promoter – Binding of repressor helps prevent RNA polymerase from binding to promoter Repressor also binds to one of two secondary operators (O2 or O3), with the DNA looped between this secondary operator and O1 – Reduces transcription, but transcription occurs at a low, basal, rate even with in the presence of the repressor HOW LAC REPRESSOR BINDS TO DNA Lac repressor is a tetramer – Dimer of dimers – Each dimer binds to palindromic operator sequence – ~17-22 base pairs of contact – Kd ~10-10 M (high affinity) O1 sequence reflects the symmetry of the repressor ~20 repressors/cell The Lac repressor tetramer binds to the main operator and O2 or O3 THE LAC OPERON IS GOVERNED BY MORE THAN REPRESSOR BINDING Availability of glucose governs expression of lactose-digesting genes via catabolite repression – When glucose is present, lactose genes are turned off – Mediated by cAMP and cAMP Receptor Protein (CRP). CRP is also called CAP for Catabolite Activator Protein WHEN GLUCOSE IS ABSENT, LAC OPERON TRANSCRIPTION IS STIMULATED BY CRP-cAMP cAMP made when [glucose] is low or absent CRP-cAMP binds near promoter – Stimulates transcription 50-fold – Bends DNA – Open complex (with RNA Pol for transcription) does not form readily without CRP-cAMP CRP-cAMP only has this effect when the Lac repressor has dissociated from DNA CRP-cAMP HOMODIMER BOUND TO DNA Bending of the DNA around the protein. TWO REQUIREMENTS FOR STRONGEST INDUCTION OF THE LAC OPERON 1) Lactose must be present to form allolactose. Allolactose binds to repressor and cause it to dissociate from operator (reducing repression of transcription) 2) Glucose must be absent or low so that [cAMP] can increase, then bind to CRP, and the CRPcAMP complex can bind near the promoter (causing activation of transcription) COMBINED EFFECTS OF GLUCOSE AND LACTOSE ON THE LAC OPERON When lactose is absent, repressor binds to the operator àinhibition (repression) of transcription of lac genes. It does not matter whether glucose is present (a) or low/absent (b) If lactose is present (c and d), the repressor dissociates from the operator. Two possibilities: - If glucose are high (c), Low cAMP levels prevent CRP-cAMP formation and DNA binding à transcription of lac genes can happen at low levels due to occasional binding of RNA Pol. - If glucose levels are low (d), cAMP levels rise and CRP-cAMP forms and binds to the DNA à activation of transcription of the lac genes at high levels. REGULATION OF THE LAC OPERON In presence of glucose ([cAMP] is low) CRP is not bound to the DNA. In the absence of lactose, allolactose is not present to bind to the repressor, so that it can bind to the DNA and block transcription. In absence of glucose ([cAMP] is high) CRP-cAMP is bound to the DNA. In the presence of lactose, allolactose binds to the repressor, preventing it from binding to the DNA. Transcription can proceed. In presence of glucose ([cAMP] is low) CRP is not bound to the DNA. In the presence of lactose, allolactose binds to the repressor, preventing it from binding to the DNA. No transcription because CRP is not bound BINDING OF PROTEINS TO DNA OFTEN INVOLVES HYDROGEN BONDING Gln/Asn can form specific H-bond Thymine-Adenine base pair Arg can form specific H-bonds with Cytosine-Guanine base pair Major groove is the right size for ahelix binding and has exposed Hbonding groups BINDING OF PROTEINS TO DNA OFTEN INVOLVES HYDROGEN BONDING Groups in DNA available for protein binding (a) Functional groups on all four base pairs that are displayed in the major and minor grooves of DNA. Hydrogen bonds acceptor (A) and donor (D) atoms are marked in blue and red respectively. Other H atoms (H) are marked purple and methyl groups (M) are marked yellow. (b) Recognition patterns for each base-pair. The greater variation in the major groove à greater discriminatory power. Examples of amino-acid-base pair Interactions in DNA-protein binding Two examples of specific amino acid residue–base pair interactions that have been observed in DNA-protein binding. REGULATORY PROTEINS HAVE DISCRETE DNA-BINDING DOMAINS/MOTIFS A few protein arrangements are used in thousands of different regulatory proteins and are hence called motifs. – Helix-turn-helix Used by Lac repressor – Zinc finger – Leucine zipper – Etc. THE HELIX-TURN-HELIX MOTIF IS COMMON IN DNA-BINDING PROTEINS The Helix-Turn-Helix motif is ~ 20 aa long – One a-helix for recognition for DNA (red in the next slide), then b-turn, then another ahelix – Sequence-specific binding due to specific contacts between the recognition helix and the major groove Four DNA-binding helix-turn-helix motifs in the Lac repressor The motif is found mainly in prokaryotes but also in eukaryotes. THE HELIX-TURN-HELIX MOTIF DNA-binding domain of the Lac repressor The helix-turn-helix motif is shown in red and orange; the DNA recognition helix is red. Entire Lac repressor (a) The DNA-binding domain of the Lac repressor bound to DNA (blue and orange). The helix-turnhelix motif is shown in dark blue and purple; the DNA recognition helix is purple. (b) Entire Lac repressor. The DNA-binding domains are light blue, and the α helices involved in tetramer formation are green. The remainder of the protein (shades of red) has the binding sites for allolactose. The allolactose-binding domains are linked to the DNA-binding domains through linker helices (yellow). THE ZINC FINGER MOTIF IS COMMON IN EUKARYOTIC TRANSCRIPTION FACTORS The zinc-binding motif is ~30 aa long “Finger” portion is a peptide loop cross-linked by Zn2+ – Zn2+ usually coordinated by 4 Cys, or 2 Cys and 2 His Interact with DNA or RNA – Binding is weak, so several zinc fingers often act in tandem Binding can range from sequence-specific to random The motif is found mainly in eukaryotes but also in prokaryotes. THE ZINC FINGER MOTIF Three zinc fingers (shades of red) of the regulatory protein Zif268, complexed with DNA (blue). Each Zn2+ coordinates with two His and two Cys residues. THE LEUCINE ZIPPER MOTIF IS COMMON IN EUKARYOTIC TRANSCRIPTION FACTORS Dimer of two amphipathic a-helices (~30 aa long each) plus a DNA-binding domain Each helix is hydrophobic on one side and hydrophilic on the other – Hydrophobic side is the contact region between the two helices ~Every seventh residue in helices is Leu Helices form a coiled coil DNA-binding domain has basic residues (Lys, Arg) to interact with polyanionic DNA The motif is found mainly in eukaryotes but also in prokaryotes. THE LEUCINE ZIPPER MOTIF Leucine zipper from the yeast activator protein GCN4. Only the “zippered” α helices (gray), derived from different subunits of the dimeric protein, are shown. The two helices wrap around each other in a coiled coil. The interacting Leu side chains (red) and the conserved residues in the DNA-binding region (orange and yellow) are shown. OTHER TYPE OF REGULATION OF GENE EXPRESSION IN BACTERIA Operon that produce the enzyme of amino-acid synthesis are regulated by “attenuation”. – Attenuation makes use of a transcription terminator site on the mRNA (the attenuator), that is sensitive to small changes in amino-acid concentrations. In the SOS system (see DNA repair) – Multiple unlinked genes, repressed by a single repressor, are induced simultaneously in response to DNA damage that triggers proteolysis of the repressor facilitated by RecA-protein. Post-transcriptional regulation of some mRNAs is mediated by small RNAs (sRNAs) that act in trans or by riboswitches (part of the RNA itself) that act in cis. Some genes are regulated by genetic recombination that move promoters relative to the genes being regulated.