How does the presence of lactose enable RNA polymerase to transcribe the lac genes quizlet?
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Lac operon in E. coli Show controls breakdown of lactose regulatory gene produces active repressor (bind operator) and block RNA pol. REGULATED AS A SINGLE UNIT. ****When lactose is available, lactose binds repressor and inactivates it => RNA pol can now transcribe. *****NO lactose---DO NOT need enzymes to metabolize lactose ******Enzymes for lactose metabolism is INDUCIBLE. Lactose induces the operon. inducible system An operon that requires an inducer to remove a repressor protein from the operator site to begin transcription of the relevant gene; also called a positive control system. INDUCIBLE system is OFF until it turns on******* cis-acting elements regulatory DNA sequences involved in eukaryotic transcription initiation can only regulate genes located on same chromosome Genetic dissection experiments mutations in different genes and use of partial diploids tryptophan operon repressible SYSTEM when trp is absent, the repressor is inactive----- when trp is present and there is enough of it, the trp binds to the repressor and activates it to stop producing Prokaryote control of gene expression -inducible system Eukaryote control of gene expression -generally lack operons Transcriptional regulation The mechanisms that collectively regulate whether or not transcription occurs. -Chromatin remodeling Chromatin remodeling A mechanism for epigenetic gene regulation by the alteration of chromatin structure. Acetylation of histones: chromatin loosens or opens for transcription so the rate for transcription increases. Deacetylation: MORE compaction and less transcription (decreased expression) Histone acetylation the attachment of acetyl groups (-COCH3) to tails of histone proteins DISRUPTS chromatin structure--the chromatin becomes less compact, and the DNA is accessible for transcription DNA methylation The addition of methyl groups (—CH3) to bases of DNA after DNA synthesis Associated with decreased gene expression Methylation occurs most often on cytosine of CG doublets in DNA.**** (C adjacent to G can get methylated) Methylation can repress transcription by binding to transcription factors of DNA. X chromosomes in female cells are heavily methylated. (inactive X) Many cancers DNA hypomethylation ("active" cells) Promoters specific region of a gene where RNA polymerase can bind and begin transcription Recognized by transcription factors (TFs) subsequently recognized by RNA pol: Core Promoter element: TATA box (25-30) Cis acting sequences Located on same chromosome as gene that it regulates; ONLY regulates genes they are adjacent to.****** DNA sequences that trans-acting factors bind to NOT involved in direct binding of RNA polymerase, but interact with TFs--possibly by altering chromatin structure and facilitate binding of RNA polymerase, or may increase the concentration of TFs near promoter (complex sex of interactions) Required for accurate regulated transcription of genes: Trans-acting factors (transcription factors) -Recognize cis acting elements of different structural genes Insulators block or insulate the effects of enhancers in a position dependent manner Examples of transcriptional regulation Hormone induced response Hormone picked up by cell, binds to receptor and acts as Transcription Factor Recognizes cis-actining HRE DNA sequence, increases transcription of a certain set of genes ex: Steroid hormones (glucocorticoid) and metallothioneine IIA gene (protects cells from toxic effects of heavy metals (ex: zinc) Insulator binding proteins When transcription factors act at a distance, the insulators (citerm-20s elements) block transcription factors from interacting with the wrong gene Prevents transcription factor from binding to the wrong gene Activators up regulate; BIND to enhancer Repressors down regulate; BIND to silencer ***Repressor gene is NOT part of operon and is expressed separately; It is a regulatory protein that has its own promoter sequence. Enhancers located on either side of gene, some distance from gene, or even within gene Silencers (down regulate): repress the level of transcription initiation Coactivator Interact with activator, alter chromatin structure forming enhancesosome Other types of regulation Posttranscriptional: Alternative splicing, RNA interference Alternative splicing Post-translational RNA modification process in which some exons are removed or joined in various combinations. Intro to control of gene expression Not all genes are expressed at all times in all situations. Many prokaryotic gene products are present continuously at low levels; these can increase as needed. In multicellular eukaryotes, differential gene expression is also essential and is at the heart of embryonic development and maintenance of the adult state. Levels of gene regulation transcriptional, post-transcriptional, translational, post-translational Bacteria and Gene expression Bacteria respond metabolically to changes in their environment (note - single-cell organisms). (MAINLY respond to environment; if environment changes, genes turn on or off) Bacteria regulate gene expression to synthesize products needed for a variety of normal cellular activity. Structural genes Protein-encoding DNA sequences that function in metabolism, biosynthesis, or cell structure **Important in maintenance Genes coding for the primary structure of an enzyme Regulatory gene Protein- or RNA-encoding DNA sequences that regulates structural genes (trans-acting)******* Help to control the expression of structural genes of the operon by increasing or decreasing their transcription. Has its own promoter and is transcribed into a short mRNA, which is translated into a small protein. Regulator protein binds to a region of the operon
Regulatory element DNA sequence that impacts expression of gene that is adjacent (cis-acting) beta-galactosidase gene encodes a protein that normally breaks down, lactose, a common sugar in milk, into two pieces, glucose and galactose Glucose= usable sugar form ALSO called lactase lactose operon In the presence of lactose, concentrations of the enzymes responsible for lactose metabolism increase rapidly from a few molecules to thousands per cell. The enzymes responsible for lactose metabolism are inducible, and lactose is the inducer. lac Z, lac Y, lac A In the lac operon, genes will be transcribed into the mRNA and eventually produce the enzymes required to break down the lactose 3 structural genes of lac operon lac Z, lac Y, lac A lacZ gene encodes β-galactosidase. - lacY gene encodes permease. -lacA gene encodes transacetylase. Lactose glucose + galactose Galactose a monosaccharide; part of the disaccharide lactose Glucose components of lac operon Regulatory region Promoter **REMEMBER repressor binds to operator******* Structural genes:
How is lactose expressed? expressed at a basal level -There is enough lactose to get into cell **Lactose binds to lactose binding site. no lactose present in lac operon lacZYA off Repressor CANNOT bind to promoter. RNA polymerase (think of it like a train and the structural genes are the track) is blocked so CANNOT synthesize RNA of structural genes; SYSTEM IS OFF; prevents transcription Allosteric reaction: repressor alters shape and DOES NOT bind to operator. Sigma factor recognizes promoter BUT RNA polymerase cannot get through. SO RNA POL recognizes promoter and synthesizes RNA from structural genes Lactose present lactose binds to repressor and inactivates the repressor protein so that transcription can occur SYSTEM IS ON so that is why lactose induces expression of operon NO binding to repressor Allosteric reaction; mutant repressor gene when Lactose absent Normally, regulatory repressor that represses expression of lac Z gene. Lac I encodes the repressor--> Decreases expression of Beta galactosidase. Mutant repressor Gene When Lactose present Genetic Dissection experiments Created mutations and looked to see what happens. Mutation to lac I gene (repressor); Loses repressor function--> Will have EXPRESSION but NO repression--> Operon ALWAYS ON SO System is always ON. Lac O operator which is the binding site for a protein (lac repressor) MUTATION of operator--> RNA POL will not bind to operator REGARDLESS if lactose present or not. DNA binding protein normally recognizes DNA sequences (operator) BUT sense operator is mutated--> DNA binding protein does NOT recognize mutated sequence Oc= constitutive operator; ALWAYS on. Genetic Proof of Operon Model Monod wanted to determine cis and trans-acting SO he created partially diploid (merozygote) bacteria for genetic dissection experiments Add genes to F factor (plasmid or extra chromosomal DNA) Determined B-galactidose activity in presence or absence of lactose Partial diploid (merozygote) a strain of bacteria containing F' factor genes too much lactose? Catabolite activating protein (CAP) exerts positive control over lac operon So if glucose is increased--> cAMP is decreased (TOO much glucose means that CAP has NO cAMP to bind to SO if you have too much, you do not want the operon to keep going, so you turn it off) No glucose?--> Increased cAMP catabolite activator protein (CAP) In order to bind to the promoter, CAP must be bound to cyclic adenosine monophosphate (cAMP). (cAMP allows CAP to bind efficiently) Binding of the CAP-cAMP complex to the promoter region increases the efficiency of binding of RNA polymerase to the promoter ****The level of cAMP is dependent on the enzyme adenyl cyclase. Glucose inhibits the activity of adenyl cyclase, which catalyzes the conversion of ATP to cAMP and thus prevents CAP from binding when glucose is present The Tryptophan (trp) Operon in E. coli Is a Repressible Gene System Tryptophan production is REPRESSIBLE (on all the time
and must be turned off by repressor SO "On until turned off.") trp= corepressor so binds to repressor and turns system off; Allosteric reaction occurs when corepressor bound to repressor AND can bind to operator and to RNA so system is turned off. Eukaryotes Tightly controlled to express required levels of gene products Prokaryotic gene regulation occurs primarily at transcription initiation. Eukaryotic gene regulation occurs at different levels. Lack operons CORE promoters must be in fixed positions enhancers and silencers speed up (enhancer) or slow down the rate of transcription (silencers) Enhancers and silencers regulate transcription of eukaryotic genes. Cis-acting transcription regulatory elements-NOT IN FIXED LOCATIONS! Transcription factors A regulatory protein that binds to DNA and affects transcription of specific genes. Transcription regulatory proteins- Trans-acting Factors Target cis-acting sites of genes regulating expression Activators increase transcription initiation. Repressors decrease transcription initiation. Multiple transcription factors bind to several different enhancers and promoter elements and fine-tune the level of transcription initiation. Transcription activators and repressors bring changes to RNA Pol II transcription. DNA looping delivers activators, repressors, and general transcription factors to promoter vicinity. Recruitment model: Enhancers and silencer elements act as donors and affect regulatory proteins at gene promoters' Activators: Proteins that bind enhancers; increase transcription Repressors: Bind silences; decrease transcription levels. coactivators proteins that increase the rate of transcription but do not directly bind to the DNA itself Interact with proteins and enable activators to make contact with promoter-bound factors Coactivators form complex "enhanceosome"**** A transcription factor may become active only when modified structurally (e.g. phosphorylation). enhanceosome a large protein complex that acts synergistically to activate transcription Binds to enhancer TATA box is recognized by TF2D Human Metallothionein IIA Gene ( Example of how a gene can be transcriptionally regulated due to interplay of: Product of hMTIIA Protein binds to heavy metals and protects cells from toxic effects. Ex: Glofish (Fish glow due to constitutive promoter so ALWAYS ON no matter what) RNA interference (RNAi) A technique to silence the expression of selected genes in nonmammalian organisms. The method uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger the breakdown of the gene's messenger RNA. Small interfering RNA---> Viral or genetically engineered dsRNA is an exact match to gene of interest; develop and inject into cytoplasm Dicer protein cuts into tiny pieces of dsRNA and separates the strands--> mRNA gets degraded and prevents translation micro RNA Naturally occurring Gets into nucleus Single stranded RNA that forms secondary structure (End up with 20 base pairs of dsRNA) BIND to RISC; Exact match--> degrades mRNA mismatch--> miRNA stays bound to mRNA and INHIBITS translation Notes from Chapter 12 Reading ... Gene expression controlled at different levels Includes: Alteration of gene structure Much of gene regulation takes place through the action of regulatory gene products that recognize and bind to regulatory elements Genes in bacterial cells Typically clustered into operons--groups of functionally related structural genes and the sequences that control their transcription. Structural genes in an operon are transcribed together as a single mRNA molecule negative control of transcription occurs when a regulatory protein called a repressor binds to DNA and shuts down transcription positive control of transcription a regulatory mechanism that starts transcription through an activator protein that activates the binding of RNA polymerase to DNA Inducible operons transcription is usually off (inhibited) and needs to be turned on (induced) The regulator protein is a repressor that binds to the operator and prevents transcription of structural genes when the inducer is present, it BINDS to the regulator, thereby making the regulator unable to bind to the operator. Transcription takes place Repressible operons transcription is normally on and needs to be turned off 1. Regulator protein is an inactive repressor, unable to bind to the operator Lac operon of E.coli negative inducible operon. In absence of lactose, repressor binds to the operator and prevents the transcription of genes that encode B-galactosidase, permease, and transacetylase in presence of lactose, some of it converts to allolactose (inducer), which binds to the repressor and makes it inactive, allowing the structural genes to be transcribed Positive control in the lac operon regulated by catabolite activator protein (CAP) When complexed with cAMP, the catabolite activator protein binds to a site in or near the promoter and stimulates the transcription of the structural genes levels of cAMP are inversely correlated with glucose, SO LOW levels of glucose stimulate transcription and HIGH levels inhibit transcription**** trp operon (E. coli) negative repressible operon that controls the biosynthesis of tryptophan in repressible operon, transcription normally turned ON and must be repressed, this is accomplished through the binding of tryptophan to the repressor, which renders the repressor active. The active repressor binds to the operator and prevents RNA polymerase from transcribing structural genes Eukaryotic vs prokaryotic gene regulation Eukaryotes= absence of operons, Presence of chromatins, and presence of nuclear membrane Eukaryotic chromatin structure Chromatin structure is directly related to the control of gene expression (represses it) Chromatin structure can be altered by chromatin remodeling complexes that reposition nucleosomes and by modifications of histone protein INCLUDING: Acetylation Methylation of DNA affects transcription Initiation of Eukaryotic Transcription Controlled by general transcription factors that assemble into the basal transcription apparatus and by transcriptional regulator proteins that stimulate or repress normal levels of transcription by binding to regulatory promoters and enhancers. Enhancers function affect transcription of distant genes Regulatory proteins bind to enhancers and interact with the basal transcription apparatus by causing the intervening DNA to loop out Insulators function limit the action of enhancers by blocking their action in a position dependent manner Coordinately Controlled Genes in Eukaryotes Respond to the same factors because they have common response elements that are stimulated by the same transcriptional activator Gene expression in eukaryotic cells Can be influenced by RNA processing and by changes in mRNA stability 5' cap, the poly (A) tail, the 5' UTR, the coding region, and sequences in the 3' UTR are important in controlling the stability of eukaryotic mRNA RNA interference Plays important role in eukaryotic gene regulation. Initiated by double stranded RNA molecules that are cleaved and processed. Small RNA molecules (siRNAs and miRNAs) combine with proteins and bind to sequences on mRNA or DNA These complexes cleave RNA, inhibit translation, affect RNA degradation, and silence transcription posttranslational modification may play a role in the regulation of gene expression Arabidopsis thaliana (A. thaliana) possess a number of characteristics that make it an ideal model genetic organism Epigenetic effects on gene expression inherited changes in gene expression NOT DUE to changes in the DNA base sequence--are frequently causes by DNA methylation and alterations in chromatin structure Epigenetic changes are STABLE but can be affected by environmental factors Many epigenetic phenotypes result from changes to chromatin structure Epigenetics effects occur through DNA methylation, histone modifications, and RNA molecules Paramutation A heritable alteration of one allele by another allele without any change in the DNA sequence. Early life experiences can produce epigenetic changes can have long lasting effects on behavior Environmental chemicals may produce epigenetic effects that are passed to later generations Phenotypic differences between genetically identical monozygotic twins may result from epigenetic effects Epigenome complete set of chromatin modifications possessed by an individual organism Operon single transcriptional unit that includes a series of structural genes, a promoter, and an operator. Comparison of gene expression in bacteria and eukaryotes Not all genes are expressed at ALL times in ALL situations Can have same genes throughout body but different genes expressed through different parts of the body. MANY prokaryotic gene products are present continuously at LOW levels; these can increase as needed. In multicellular eukaryotes, differential gene expression is also essential and is at the heart of embryonic development and maintenance of the adult state.
Tricks for Gene activity problems (Presence/absence of lactose) Z-: NEVER make functional B-galactosidase SO B-gal activity with or without lactose present I-: Mutant repressor, ALWAYS make B-galactosidase Oc= constitutive operator; Repressor cannot bind so never turned off; ALWAYS produces nonfunctional B-gal. Ic: super-repressor; CANNOT be stopped from binding to operator EXCEPT constitutive operator can stop super repressor.---> ALWAYS binds to operator so NO product Lac I vs. Lac O Lac I works in trans Difference between cis vs trans-acting elements By using different combinations of mutations on the bacterial and plasmid DNA, Jacob and Monod determined that some parts of the lac operon are cis acting (able to control the expression of genes only when on the same piece of DNA), whereas other parts are trans acting (able to control the expression of genes on other DNA molecules). Understanding cis and trans-acting element For E. coli strains with the lac genotypes given below, use a plus sign (+) to indicate the synthesis of β-galactosidase and permease and a minus sign (−) to indicate no synthesis of the enzymes. How does the presence of lactose enable RNA polymerase to transcribe the lac genes?When lactose is bound to lacI, the shape of the protein changes in a way that prevents it from binding to the operator. Therefore, in the presence of lactose, RNA polymerase is able to bind to the promoter and transcribe the lac operon, leading to a moderate level of expression of the lacZ, lacY, and lacA genes.
What is RNA polymerase doing on the lac operon in the presence of lactose quizlet?The operon is induced when lactose molecules bind to the repressor protein. As a result, the repressor protein loses its shape and falls off of the operator region. Therefore, RNA polymerase can continue transcribing the rest of the operon.
How does the presence of lactose cause the lac operon to be transcribed quizlet?How does the presence of lactose in the cell influence the transcription of the lac operon ? - When lactose is present, the lac protein does not bind to the operator site and the transcription can occur. - when lactose is present, transcription of the lac operon is high.
How does lactose cause the lac operon to turn on?How does lactose cause the lac operon to turn on? Lactose molecules bind to the repressor protein, causing it to change shape so that the repressor releases the operator. This allows RNA polymerase to bind to the promoter and transcribe the genes of the operon.
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