Genetics Vocabulary

The Pajama Experiment & Lac Operon

• The Pajama Experiment: Evidence that LacI encodes a negative regulator of Lac.
  • Production of LacZ stops after 2 hours unless an inducer is added.
  • Some time is needed for the production of LacI.
  • LacI is a repressor that is inactivated by an inducer.
  • Wild type is lacI+ and recessive is Lac-.
  • Initial burst of beta-galactosidase occurs because LacI hasn't accumulated enough to exert its role on lac Z.
  • After 3 hours, there's a reduction over time of beta-galactosidase to almost nothing.
  • In the presence of IPTG, LacI can't perform its role as a repressor, and beta-galactosidase synthesis continues.
• Transcription Regulation:
  • If lacI+ and lac promoter and structural genes are present with lactose, there is production of beta-galactosidase and permease. Without lactose, there is no production.
  • A mutation in the structural gene results in a non-functional product because beta-galactosidase is not produced.
  • A mutation in permease results in a non-functional product because permease is not produced.

Mutations in lacO and lacI

• LacI has different domains that, when mutated independently, yield different phenotypes.
  • Various activities of LacI (DNA binding, inducer binding) are genetically separable.
  • In a product, lac repressor, and in the presence of an inducer, there's a reduction of affinity for the lac repressor for lac O.
  • If a mutation is introduced in the lac O DNA sequence, the lac repressor no longer recognizes the site effectively, leading to no more repression, so lac production is constitutive; called lac Oc mutations.
  • Mutations in the DNA binding domain in LacI lead to not being able to recognize effectively lac O with high affinity, get mutation called lac I- which leads to constitutive synthesis of lac operon.
  • The lacI protein is a homotetramer; each subunit associates with 3 other subunits; each subunit has a DNA binding region and an inducer binding region.
  • A repressor protein mutation blocks binding to the inducer, preventing the formation of the inducer-repressor complex.
  • A mutant repressor protein binds to the operator, preventing transcription; called super-repressor mutation; this is IS mutation.
• Constitutive vs. Uninducible:
  • Constitutive transcription can be due to a mutation in lacOc or lacI- in the DNA binding domain.
  • Uninducible can be due to lac IS.
  • No effective transcription due to lacP- mutation; lac promoter mutation in -10 or -35 box results in this.

Lac Repressor

• lacI is a homotetramer: 4 subunits interact.
• All 4 DNA binding domains of LacI must be engaged on the DNA (lacO) for high affinity binding.
• Binding of an inducer reduces affinity for lacO through allosteric effects.
• Binding of an inducer leads to a conformational change of the whole molecule, including the DNA binding domain.

Footprinting

• Lac Repressor Binding to Operator:
  • Unprotected DNA is digested (cut) by DNase I.
  • Binding sites for LacI and RNA polymerase overlap; they cannot bind at the same time. This is called steric hindrance.
  • Experiment:
    • Radioactively label the DNA, mix with proteins, and then use DNAse 1 that cuts naked DNA.
    • Lane 1: Naked DNA, which cuts in absence of protein.
    • Lane 2: DNAse cuts when RNA polymerase is bound to segment, protecting a large region that contains the whole promoter region.
    • Lane 3: Just the repressor, showing what the repressor protects against DNase 1 cleavage.
  • RNA polymerase can’t be bound to the promoter if the repressor is bound to the operator; mutually exclusive.
• Lac Repressor Binding to Operator:
  • There are 2 operator sites.
  • lacO1 contains an inverted repeat sequence, leading to the binding of 2 of the subunits of lacO.
  • lacI DNA binding domains are from diff subunits in the homotetramer.

LacO3 & Lambda Bacteriophage Genome

• LacO3: Two other subunits bind LacO3, which is further upstream of the promoter.
  • Binding leads to the formation of a knot by DNA looping that further prevents RNA polymerase from binding.
  • lacO1 makes steric hindrance.
  • lacO3 overlaps with the CRP/CAP site, and there is a CAP site overlapping with the operator.
  • When CRP binds to cAMP, it dimerizes, binds to the CRP-cAMP binding site, and recruits RNA polymerase.
  • There’s physical contact between CRP and RNA polymerase, allowing full activation of the lac promoter in the absence of glucose.
  • In the presence of glucose, there is no production of cAMP, and the complex doesn’t bind the CAP site.
• Lambda Bacteriophage Genome:
  • As lambda infects the host, there are molecular interactions between proteins called C1 and CRO.
    • If Cro wins, lytic cycle; if C1 wins, lysogenic cycle.
  • 49700 base pairs.
  • There are proteins that code for the head subunits and others for the tail subunit.
  • c1 and Cro are tiny parts of the genome.
  • C1 encodes a protein called lambda repressor, and Cro encodes cro.
  • Gene Q is a regulator of late genes; if Q is transcribed and translated, then there will be lysis.
    • Q is transcribed and translated if Cro gets expressed; if C1 gets expressed, Cro gets shut off and leads to lysogeny.
  • Lambda isn’t covalently closed; it's circularized through bp, but there’s no covalent bond between the bottom and top strands.

The Genetic Switch

• It can integrate through enzymatic integration into the E. coli genome at a specific place between genes galK and BioA.
• The Genetic Switch:
  • Lysogeny: C1 expression.
  • Lysis: Cro expression.
  • C1 is expressed from a promoter called PRM; only protein made by prophage and it represses.
  • Cro is expressed from promoter called Pr.
  • During lysis, Pr is on, Prm is off.
  • Cro: Or3 > Or2 = Or1; represses C1; induces excision and lytic cycle.
  • C1: I repressor OR1 > OR2 > OR3 is affinity establishes and maintains lysogeny.
  • Cro turns off C1, PR is active and transcribes Q, activator of late gene transcription.
  • C1 binds right on top of PR, blocking Cro; vice versa is true.
  • C1 binds as a dimer to itself; can recruit itself and promote its own binding to OR2.
    • When C1 binds to Or2, it actually favors the recruitment of RNA polymerase to PRM.
  • C1 turns off Cro and activates its own transcription.
  • C1 can then bind to OR3 last, and when it binds to OR3, it shuts off its own transcription.
  • C1 autoregulates its amounts.

Termination of Lysogeny

• C1 is composed of 2 separate domains.
  • One domain is the DNA binding domain that as a dimer can bind the operator sequences.
  • The other domain is able to interact and form a dimer that binds Or1, but C1 can recruit another dimer to OR2; this activates its own transcription.
• Excision and lysis can be induced by UV: this leads to cleavage of C1 by RecA.
  • RecA has proteolytic activity.
  • UV activates the protease RecA, which cleaves C1 in the domain that joins the N and C terminal of C1; in doing so, C1 can no longer form a dimer and bind DNA effectively.
  • C1 is taken off the DNA; the sites are freed up; OR1 is no longer bound by C1.
  • RNA polymerase can initiate transcription from Pr and transcribe Cro, which then represses C1, shuts off transcription of C1, and Q is transcribed, leading to the lytic cycle.

Overview of the Genetic Switch

• How is lysogeny versus lysis determined?
  • During infection: the outcome depends on who wins.
    • This depends on growth conditions; under good conditions, c1 is degraded, and lysis is favored.
    • Under good growth conditions, a protein called c2 is degraded, and the degradation leads to the activation of Pr and deactivation of Prm.
    • C2 is necessary for the initial burst of C1 production.
    • Poor growth conditions: lysogeny is favored, and c1 wins.

Trp Operon

• This is an anabolic pathway, repressed by its end product: Trp.
• Leader sequence is transcribed and is important in regulating transcription elongation on the RNA; at the beginning of the transcription unit.
• The regulatory region has: Ptrp-trpO (operator) and trpL: leader region/sequence that itself contains the attenuator region.
• The Trp operon has 5 genes; there is a promoter, an operator, and the leader sequence.

Trp Amino Acid & TrpR Mutants

• Trp amino acid is a co-repressor: end product is negative feedback on the amount of tryptophan.
• Trp repressor binds the operator under high tryptophan levels; it acts as a corepressor, binds the repressor, and sees no transcription.
• The binding of the repressor to trpO requires association with a co-repressor.
• TrpR Mutants:
  • De-repression is only partial.
  • The feedback loop only counts for 33% of the repression.
  • Something else is repressing the operon.
• Leader:
  • The leader has an additional role after RNA polymerase recruitment.
  • The L region is upstream of the first structural gene.
  • This leader sequence contains an open reading frame with a start and stop codon.
• 1-2 and 3-4 presents a terminator of transcription.
  • This structure on the RNA depends on Trp availability.
  • This structure will form under abundant Trp conditions.
  • trpL contains 4 small sequences with partial base pairing capacity: 1-2 and 3-4 or 2-3.
    • If 2 forms a hairpin with 3, 1 and 4 are freed up and left alone.

Trp Operon Details

• The 3-4 stem loop presents a Rho-independent transcription terminator; this is a stem loop followed by a stretch of UUs.
  • When 1 pairs with 2 and 3 pairs with 4, that terminates transcription right away.
  • When 2 pairs with 3, this motif isn’t formed, and transcription elongation can proceed.
  • What pairs with what depends on the amount of tryptophan.
  • When tryptophan is abundant, 3 pairs with 4, and transcription termination occurs, and the operon isn’t transcribed.
  • Under scarce tryptophan conditions, 2 pairs with 3, and transcription elongation proceeds.
• How tryptophan regulates these structures has to do with the presence of tryptophan codons present within sequence 1.
• Trp Operon - Tryptophan Abundance:
  • The trpL region also contains a reading frame for 14 amino acids, including 2 sequential codons for Trp.
  • Translation proceeds all the way to the stop codon.
  • Region 2 is unavailable to pair with 3, and 3 can pair with 4; production of a stem-loop structure followed by UUUUU.
  • The ribosome sits on region 2 and allows 3 and 4 to form a transcription termination structure.
• Trp Operon - Poor Conditions:
  • Under poor tryptophan conditions, the ribosome stalls because there are 2 contiguous tryptophan codons on region 1.
  • If no tryptophan is available, very few aminoacyl tryptophan tRNAs are available.
  • Translation stalls on 1, and 2 and 3 will pair, and then the transcription terminator can’t form, and therefore transcription elongation proceeds through the operon.
• Trp Mutations:
  • The importance of stem loops can be genetically shown by disrupting them by mutation.
  • Base pairing can be disrupted by mutation.
  • Reduced ability to sense the level of Trp.
  • As a result, if in low or high Trp conditions, depending on which stem loop you mutate, you will be constantly terminating transcription or constitutively translating the operon no matter the levels of Trp.
  • The formation of stem loops is important to this system.

Trp Operon

• There are other amino acid biosynthesis operons that work the same way, but in their leader sequence, they don’t have tryptophan codons; ex: for his operon, his codons, etc.
• In these operons, the level of the AA’s corresponding to them is sensed.

Chapter 13 - Regulation of Gene Expression in Eukaryotes

• Overview of gene regulation mechanisms in eukaryotes: All diff levels going from DNA to functional proteins, looking at how the final function of a protein is regulated.
• Transcriptional regulation in eukaryotes:
  • DNA in eukaryotes is packed into nucleosomes.
    • In nucleosomes, there are proteins called histones that interact with DNA.
  • For transcription to occur, the DNA has to be opened.
  • Chromatin status is different depending on the degree of packing: euchromatin (active) vs. heterochromatin (silent).
• Epigenetic modifications - adding chemical groups that affect chromatin interaction with DNA.
• There are different regions in DNA that act as regulatory regions.
  • Cis-acting regulatory regions: enhancer, proximal element, and core promoter (TATA box).
  • Trans-acting regulatory proteins: transcription factors, TATA binding proteins (TBP), general transcription factors (GFT).
  • The binding of regulatory proteins to the sequences allows for transcription to be initiated with the binding of RNA polymerase II.
  • To be transcribed, you need at least enhancers and promoters.
• Cis-acting regulatory sequences bind trans-acting regulatory proteins to control eukaryotic transcription.
  • Activator proteins: bind regulatory sequences to stimulate transcription.
  • Repressor proteins: bind other sequences to hinder transcription.
  • Regulatory proteins are often found in large complexes in eukaryotes, unlike in bacteria.
• Individual transcription factors may regulate tens to hundreds of target genes.
  • A combo of diff activators and repressor proteins work together.
  • These proteins rarely work by themselves; they have different domains that allow them to interact with DNA and also other proteins.
• Cis-Acting Regulatory Elements:
  • Promoters and enhancers.
  • DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression.
    • Cis: same side as the gene they regulate.
    • Can be upstream or downstream of the coding sequence; here, there are 3 activators and 1 inhibitory.

Cis-Element Regulatory Structures in Eukaryotes

• Blue arrow is the coding region.
• The top is for saccharomyces (unicellular), and the bottom is a multicellular eukaryote.
• In red is the core promoter, the TATA box.
• On top, there are 4 elements that are upstream from the promoter.
• Another gene on the top may or may not be regulated by the same enhancers.
• The bottom shows that sometimes regulatory sequences may even be downstream and even in an intron area.
• Some enhancers could be located at a distance from the gene.

Overview of Transcriptional Regulatory Interactions in Eukaryotes

• Different sets of regulatory DNA sequences are commonly involved in eukaryotic gene regulation.
  • The core promoter region, containing the TATA box and other sequences, is immediately adjacent to the start of transcription; these bind RNA polymerase II and its associated general transcription factors (GTFs).
  • Upstream of the core promoter region are various proximal elements that also regulate genes.
• Enhancer and silencer sequences:
  • At greater distances from the core promoter are the enhancer and silencer sequences; these bind regulatory proteins and interact with proteins bound to other promoter segments.
  • May be close to or very far upstream or downstream from the genes they regulate; may even be within genes they regulate.
  • Silencer are negative enhancers.
• Cis-acting reg sequences and trans-acting proteins:
  • All regulatory regions previously described contain cis-acting regulatory sequences, which regulate the transcription of genes on the same chromosome as the sequences.
  • RNA polymerase II and various GTFs bind the core promoter; these trans-acting regulatory proteins can bind to their target sequences on any chromosome.
• At enhancers, aggregations of multiple proteins form large complexes called enhanceosomes.

Enhancers

• Activate transcription in cooperation with promoters.
• Enhanceosomes bend DNA into loops, and they are multiprotein complexes (also called mediator complex).
  • The loops allow enhanceosome proteins to interact with RNA polymerase and transcription factors at the core promoter and proximal promoter elements.
  • Loops may be small or large based on the distance between the enhancer sequence and the gene it regulates.
  • Enhancers bring the activator proteins and coactivator proteins to the RNA polymerase.
• Integration and modularity of eukaryotic regulatory sequences:
  • Enhancers and silencers typically contain binding sites (modules) for a number of transcription factors.
  • Modules allow enhancers and silencers to integrate the activities of different transcription factors to produce different outputs.
• Pioneer factors are first to facilitate the binding of additional transcription factors.
• Eukaryotic enhancer and silencer module:
  • Occasionally, the same sequence can act as an enhancer or silencer, depending on which regulatory proteins are present and bind to the sequence.
  • If only activators are on and no repressors, the gene is on; if only repressors are on and no activators, the gene is off; may have both activators and repressors on.

Protein Binding & Sonic Hedgehog Gene

• 2 proteins might bind