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Define regulator gene.
It codes for a protein that controls transcription by binding to a particular site on DNA
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Define negative control
A trans-acting repressor protein binds to the cis-acting operator to turn off transcription.
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Define positive regulation
A trans-acting protein binds to the cis-acting site in order for RNA pol to initiate transcription at the promoter
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Define operon
A unit of bacterial gene expression and regulation, including structural genes and control elements in DAN recognized by regulator gene products
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Define trans-acting factor
A gene product that can diffuse to act on a target sequence
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Define cis-acting sequence
A DNA sequence that function only as a sequence and affects only the DNA to which it is physically linked
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Define structural gene
A gene that codes for a protein product
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Define inducible
A genes ability to express due to the appearance of a protein product
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Define repressible
A genes ability to be controlled by the amount of product made by the protein
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Define corepressor
A molecule that prevents the production of enzymes that are able to synthesize them
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What are the combinations of gene regulation
- - inducible
- - repressible
- + inducible
- + repressible
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What is the lac operon
- 1. lacI - encodes the lac repressor (diffusable) and has its own promoter and terminator
- 2. Operator - lies between the promoter and lacZYA
- 3. lacZYA - encodes polycistronic mRNA for products that digest B-galactosides (e.g. lactose)
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How does the lac repressor work
- 1. The operator overlaps the promoter
- 2. The repressor binds at the operator
- 3. Repressor binding prevents RNA pol from initiating ranscription at the promoter
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What are the essential features of B-galactoside induction
- 1. Addition of the inducer results in rapid induction of lac mRNA
- 2. This is followed by enzyme synthesis
- 3. Removal of the inducer is followed by rapid cessation of synthesis
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Why is the lac gene expressed in the absence of an inducer
lac repressor is bound to the operator preventing RNA pol from initiating transcription
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How do B-galactosides induce transcription of the lac operon
- 1. lac repressor is allosteric
- 2. The inducer binds the lac repressor and changes the DNA binding site on the repressor such that it has a lower affinity for the DNA lac operator
- 3. RNA pol binds the promoter and transcribes the operon
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Why are operator mutations constitutive
- 1. The operator can't bind the repressor
- 2. RNA pol has unrestricted access to the promoter
- 3. Oc are cis-acting because they affect only the contiguous set of structural genes
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Why is lacI- a constitutive mutation
- 1. lacI- mutations render the lac repressor inactive
- 2. Inactive lac repressor can't bind the operator
- 3. The operon is constitutively expressed
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Explain the dominant negative behavior of the lacI-d mutation
- 1. A lacI-d mutant gene makes a monmer that has a damaged DNA binding site
- 2. When present in the same cell with the WT gene, multimeric repressors are assembled at random from both types of monomeric subunits
- 3. It only requires one of the subunits of the multimer to be of the lacI-d type to prevent repressor from binding the operator DNA
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What is the general structure of lac repressor monomer
- 1. Helix-Turn-Helix - DNA binding domain
- 2. Core - contains inducer binding site and multimerization binding sites
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What is the general structure of the lac repressor tetramer
- 1. Consists of 2 dimers
- 2. Dimers are held together by interactions between the core subdomains 1 and 2
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What are the 2 types of mutations in lac repressor
- 1. lacI- - recessive and can't repress
- 2. lacIs - dominant; either can't bind to DNA or can't be bound by inducer
- 3. lacI-d - dominant negative; cna't bind DNA
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How does the repressor recognize the operator
The operator is palindromic
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How is lac repressor regulated by an allosteric change in conformation
- 1. The DNA binding domain of lac repressor has a conformation that makes it easy to bind to successive major groves of the operator DNA
- 2. When the inducer binds lac repressor, it changes the orientation of the DNA binding domain and makes it harder for lac repressor to bind operator DNA
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What happens if both dimers in a repressor tetramer binds to DNA
- 1. The DNA between the two binding sites forms a loop
- 2. This is important to full repression
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How does comparing equilibruim constants for repressor/operator binding with inducer present and with inducer absent.
- 1. The equilibrium constat for repressor/operator binding with inducer absent is 107
- 2. The equilibrium constat for repressor/operator binding with inducer present is 104
- 3. Repressor will bind 1000 times more easily when inducer is absent
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How does the operator compete with low affinity sites to bind repressor
- 1. Active repressor stays bound to operator
- 2. Repressor inactivated by inducer binds random low affinity DNA sites
- 3. When inducer leaves, repressor becomes active and is attracted by operator
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How does cAMP work as an inducer in lac operon regulation
- 1. cAMP activates inactive CRP
- 2. Active CRP binds the lac promoter and assists RNA pol in initiating transcription
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How does glucose inhibit transcrition of operons that require CRP
- 1. cAMP is synthesized by adenylate cyclase
- 2. Glucose represses adenylate cyclase activity
- 3. No cAMP = inactive CRP = no transcription
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What is the organization of the trp operon
Promoter-Operator-Leader-Attenuator-trpEDCBA-terminator-space-terminator'
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How is the trp operon negatively controlled
- 1. It is controlled by the level of its product, tryptophan (autoregulation)
- 2. Tryptophan activates an inactive repressor encoded by trpR
- 3. The repressor will act on all 3 operator loci
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What is attenuation
The regulation of bacerial operons by controlling termination via changes in RNA secondary structure due to ribosome movement
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How is the trp operon controlled by attenuation
- 1. An attenuator controls the progression of RNA pol into the trp genes
- 2. RNA pol initiates at the promoter and then proceeds before pausing at the attenuator
- 3. In the absence of Trp, RNA pol continues transcribing the trp structural genes
- 4. In the presence of Trp, RNA pol most likely terminates transcription
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What is a leader peptide
- 1. The expression of a short sequence that is located between the operator and the attenuator on the trp operon
- 2. The structure of mRNA at the attenuator depends on whether this reading frame is translated
- 3. In the presence of Trp-tRNA, the leader is translated to the leader peptide, and the attenuator is able to from the hairpin that causes termination
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What conformation must the mRNA trp leader region exist in order to terminate transcription
Regions 3 and 4 must be paired
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How does the position of the ribosome on the mRNA determine the mRNA structure during trp transcription
- 1. In the absence of Trp, the ribosome stalls at the leader sequence
- 2. Regions 2:3 pair
- 3. RNA pol continues to the trp genes
- 4. In the presence of Trp, the ribosome translates the leader sequence and disrupts the 2:3 pairing
- 5. Regions 3:4 pair forming a hairpin
- 6. Termination
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How can translation be regulated by a regulator protein
- 1. A regulator protein binds to the ribosome binding site
- 2. Ribosome can't bind to mRNA
- 3. No translation
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How is r-Protein synthesis controlled by autoregulation
The product of a first initiation site changes the mRNA secondary structure such that the second initiation site is blocked and translation stops
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How is translation of the r-protein operon autogenously controlled
- 1. When rRNA is available, the r-proteins associate with it and translation of mRN continues
- 2. When no rRNA is available, r-proteins accumulate and begins to bind to mRNA and prevents translation
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