Gene Regulation in Prokaryotes

  1. Some bacteria are able to produce chemicals that produce bioluminescence have a _______ relationship with some animals, for example when they illuminate the squid they live on. This helps the squid evade certain predators.

    *We also use bacteria in symbiotic relationships as well, for example: our digestive system would not work properly without a lot of the bacteria that inhabit it. Therefore, prok gene regulation isn’t just interesting, it is also very important to our health
  2. Prokaryotes can terminate transcription when the mRNA forms a ______ due to complementary base pairing. Transcription can also be terminated when an mRNA sequence is recognized by the protein ____, which then signals the polymerase to end transcription
    • hairpin
    • Rho
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  3. 5 general characteristics of proks and 3 translational characteristics of proks mRNAs

    *see page 282 to compare to euks
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  4. Three factors of transcriptional control in Proks
    Three factors of postranscriptional control in Proks
    • Binding of RNA polymerase to the promoter (primary mechanism)
    • Transitioning between initiation and elongation
    • Release of mRNA at termination

    • Stability of mRNA
    • Efficiency of translation
    • Stability of polypeptide
  5. Researchers first delineated basic principles of gene regulation through studies of various metabolic pathways in E. coli. Many of these pathways are _______ _______ (define + ex)
    • catabolic pathways: complicated molecules are broken down for the use of the cell 
    • ex: pathways that break down sugars to provide cells with energy and carbon atoms
  6. Other pathways in E. coli (and cells in general) are ______ ________ (define + ex)
    anabolic pathways: allow cells to construct end product molecules they need, such as amino acids and nucleotides, from simpler constituents
  7. The underlying logic cells must follow to regulate catabolic and anabolic pathways is entirely different. Catabolic pathways demand _______ ________ (explain)
    inducible regulation, meaning the pathway should be turned on, that is, induced only when the complex molecules to be broken down (catabolites) are present in the cell's environment
  8. Why would it be a waste of resources if the catabolites were not present?
    The cell would waste resources in synthesizing the enzymes needed to break down a particular sugar and that sugar (the catabolite) would not be available to cell
  9. In contrast, anabolic pathways require _______ regulation. Explain
    repressible regulation, meaning the pathway should be turned on only when the cell does not have enough of the needed end product
  10. In repressible regulation, if the end product is present in sufficient quantities, the pathway should be turned _____. (why?)
    off (repressed), so the cell does not waste resources trying to make molecules that it already has
  11. Proliferating E. coli can use any one of several sugars as a source of ______ and _____. One of these is lactose, a complex sugar composed of two monosaccharides: _______ and _______. A membrane protein, _____ ______, transports lactose in the medium into the E. coli cell
    • carbon and energy
    • glucose and galactose
    • Lac permease
  12. Once inside of the cell, the enzyme __________ splits the lactose into ________ and ________. Note that this is a ________ pathway that breaks down lactose into simpler subcomponents
    Image Upload 3
    • β-galactosidase
    • galactose and glucose
    • catabolic
  13. The two proteins ___ ______ and __________, both required for lactose utilization, are present at very _____ levels in cells grown without lactose. The cell has no need for either of these proteins if _______ is not present. The addition of lactose to the bacterial medium causes a 1000 fold _______ in the production of these proteins
    • Lac permease and β-galactosidase 
    • low
    • lactose
    • increase
  14. The process by which a specific molecule stimulates synthesis of a given protein is known as _______. The molecule responsible for stimulating production of the protein is called the _______. In the regulatory system under consideration, lactose modified to a derivative known as _______ is the inducer of the genes for lactose utilization
    • induction
    • inducer
    • allolactose
  15. Jacques Monod was a catalyst for research on the regulation of lactose utilization. Results from many genetic studies led Monod and his close collaborator Francois Jacob to propose a model of gene regulation known as the _____ _____ (what did it suggest?)
    operon theory: suggested that a single signal can simultaneously regulate the expression of several genes that are clustered together on a chromosome and are involved in the same process
  16. The reasoning behind operon theory 
    Define operon
    • They figured because these genes form a cluster, they can be transcribed together into a single mRNA, and thus anything that regulates the transcription of this mRNA will affect all the genes in the cluster. 
    • Operon: unit of DNA composed of specific genes, a promoter and or operator, that acts in unison to regulate the response of the structural genes to environmental changes
  17. In the fig for operon theory, three structural genes (name them) encoding proteins needed for _____ ______, together with two regulator elements, the ______ and the ________ make up the lac operon (define)
    • lacZ, lacY and lacA
    • lactose utilization 
    • promoter (P)
    • operator (σ) 
    • lac operon: a single DNA unit enabling the simultaneous regulation of the three structural genes in response to environmental changes
  18. Molecules that interact with the operon include the ______, which binds to the operon's operator, and the _______ (allactose), which when present, binds to the repressor and prevents it from binding to the operator. The repressor is an ______ ______ (define)
    • repressor 
    • inducer
    • allosteric protein:a protein that undergoes a reversible change in conformation when bound to another molecule (in this case, allactose)
  19. The lactose operon in E. coli 
    a)The players (5-story)
    b)Repression in the absence of _____, the repressor binds to the DNA of the ______, and this binding prevents transcription. The repressor thus as a _______ regulatory element 
    c)Induction (2-story)
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    • a)pg 519
    • b)lactose/operator/negative
    • c)pg 519
  20. Lac- mutants are bacterial cells unable to utilize ______. Using complementation analysis of a large umber of Lac- mutants, the researchers showed that the cells' inability to break down _______ resulted from mutation in which two genes (also what does each encode)?
    They also discovered a third lac gene, lacA, which encodes a _________ enzyme that adds an _____ group to lactose and other sugars
    • lactose
    • lactose
    • lacZ, which encodes β-galactosidase 
    • lacY, which encodes Lac permease
    • transacetylase enzyme
    • acetyl group (CH3CO)
  21. Genetic mapping showed that the three genes appear on the bacterial chromosome in a tightly linked ______, in the order lacZ-lacY-lacA. Why do most studies of lactose utilization  focus only on lacZ and lacY?
    • cluster
    • because the lacA gene product is not required for the breakdown of lactose
  22. Mutations in a gene called lacI produce ______ mutants that synthesize ____________ and _____ ______ even in the absence of ________. ________ mutants synthesize certain enzymes all the time, irrespective of environmental conditions. 
    Where is lacI gene located?
    • constitutive mutants 
    • β-galactosidase and lac permease
    • lactose
    • Constitutive mutants
    • located near but not within the lac operon
  23. The existence of these constitutive mutants suggested that lacI encodes a _______ regulator, or ________. Cells would need such a _______ to prevent expression of lacY and lacZ in the absence of _______. In constitutive mutants, however, a mutation in the lacI gene generates a defect in the _______ protein that prevents it from carrying out this ________ regulatory function
    • negative regulator, or repressor
    • repressor 
    • inducer 
    • repressor 
    • negative
  24. lacI indeed encodes this hypothetical _______ regulator of the lac genes. Matings in the which the chromosomal DNA of an Hfr donor cell is transferred into an F- recipient cell served as the basis of the PaJaMo study. How does it work (3-story)
    Image Upload 7
    • negative regulator 
    • The researchers transferred the lacI+ and lacZ+ alleles into a bacterial cell devoid of LacI and LacZ proteins in a medium containing no lactose
    • Shortly after the transfer of the lacI+ and lacZ+ genes, the researchers detected synthesis of β-galactosidase
    • Within about an hour, however, this synthesis stopped
  25. How did Pardee, Jacob, and Monod interpret their results? (3-story)
    • When the donor DNA is first transferred to the recipient, there is no repressor (LacI protein) in the recipient cell's cytoplasm because the recipient cell's chromosome is lacI- 
    • In the absence of repressor, the lacY and lacZ genes are expressed
    • Over time, the recipient cell begins to make the Lac repressor protein from the lacI+ gene introduced by the mating, so expression is again repressed
  26. On the basis of these experiments, Monod and company proposed that the repressor protein prevents further _______ of lacY and lacZ by binding to a hypothetical ________ site (explain)
    • transcription
    • operator site: a DNA sequence near the promoter of the lactose-utilization genes
  27. They suggested that the binding of repressor to this operator site blocks the ______, when does this binding occur and what were their further predictions?
    • promoter
    • When lactose is not present in the medium 
    • They further predicted that although some lacI- alleles would be null mutations that could not make any protein, other lacI- mutations would make a form of the repressor protein that was unable to bind to the operator
  28. In the final step of the PaJaMo experiment, the researchers added lactose (the _______ of the inducer) to the culture medium. With this addition, the synthesis of _________ resumed. What was their interpretation of this result?
    • precursor 
    • β-galactosidase 

    • The inducer binds to the wild type repressor
    • This binding changes the shape of the repressor protein so that it can no longer bind to DNA 
    • When the inducer is removed from the environment, the repressor, free of inducer, reverts to its DNA-bindable shape
  29. The binding of inducer to repressor thus causes an ________ effects that abolishes the _______'s ability to bind the ________. In this sequence of events, the inducer is an _______ molecule that releases repression without itself _______ to the DNA
    • allosteric effect
    • repressor's
    • operator 
    • effector
    • binding
  30. While lacI- mutations in the repressor gene can erase repressor activity, mutations that alter the specific ______ ______ of the operator recognized by the repressor can have the same effect. When mutations change the ______ ______ of the operator, the _______ is unable to recognize and bind to the site; the resulting phenotype is the _______ _______ of the lactose-utilization proteins
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    • nucleotide sequence
    • nucleotide sequence 
    • repressor
    • constitutive synthesis 
    • *Researchers have isolated constitutive mutants whose genetic defects map to the lac operator site, which is adjacent to the lacZ and lacY genes. They call the constitutive operator DNA alterations oc mutations
  31. If binding of the inducer to the repressor protein prevents the repressor from binding to the operator, what outcome would you predict for mutations that prevent the repressor from interacting with the inducer?
    Clearly, you would expect that such mutations would result in cells that could not turn on the operon, even when inducer was added to the medium
  32. Researchers eventually isolated such noninducible mutations in the repressor gene and designated them as lacIs, or _________ mutations. The lacIs mutants, although they cannot bind inducer, can still bind to _____ and repress transcription of the ______. This repressed state is independent of the presence or absence of ______ or _______
    • superrepressor mutation 
    • DNA
    • operon 
    • lactose or allolactose 
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  33. Elements that act in trans can ______ through the ______ and act at target ____ sites on any ____ molecule in the cell. Elements that act in cis can influence only the expression of _______ _____ on the _____ DNA molecule
    • diffuse 
    • cytoplasm
    • DNA
    • DNA 
    • adjacent genes
    • same
    • *Studies of merodiploids (partial diploids) in which a second copy of the lac genes was introduced helped distinguish mutations in the operator site (oc), which act in cis, from mutations in lacI, which encodes a protein that acts in trans
  34. In one experiment, Monod and colleagues used a lacI- lacZ+ lacY- bacterial strain that was constitutive for β-galactosidase production because it could not synthesize _______. The introduction of an F' (lacI+ lacZ- lacY+) plasmid into this strain created a ________ that was phenotypically ____ ____ with respect to both ______ and ______ expression: Both lacZ+ and lacY+ were repressible in the absence of ______ and were _______ in its presence
    • repressor
    • merodiploid 
    • wild type 
    • β-galactosidase and  permease
    • lactose
    • inducible 
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  35. The wild-type phenotype of the merodiploid indicated that lacI+ is _______ to lacI-. Moreover, what does the inducibility of not only lacY+ (on the plasmid with lacI+), but also laZ+ (on the bacterial chromosome) mean?
    Image Upload 12
    • dominant 
    • LacI protein produced from the lacI+ gene on the plasmid can bind to the operator on its own chromosome and also to the operator on the bacterial chromosome
  36. Thus, the product of the lacI gene is a _____-acting protein able to ______ inside the cell and bind to any _______ site it encounters, regardless of the operator's ________ location
    • trans-acting
    • diffuse
    • operator site
    • chromosomal
  37. In a second experiment, the introduction of a lacIs plasmid into lacI+ strain of bacteria that was orginally both _______ and _______ created bacteria that were still _______ but were no longer _______. Why did this effect occur?
    • repressible and inducible
    • repressible 
    • inducible 
    • because the mutant LacIs repressor, while still able to bind to the operator, could no longer bind inducer
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  38. The allele encoding the ________ ________ was dominant to the _____-_____ repressor allele (why?)
    • noninducible superrepressor 
    • wild-type
    • because after a while, the mutant repressor, unable to bind inducer, occupied all the operator sites and blocked all lac gene transcription in the cell
  39. In a third set of experiments, the researchers began with lacI+ oc lacZ+ lacY- bacteria that were constitutive for β-galactosidase synthesis. (why?)
    • because the wild type repressor they produced could not bind to the altered operator 
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  40. Introduction of an F' (lacI+ o+ lacZ- lacY+) plasmid did not change this state of affairs, the cells remained _______ for _______ production, although they now were inducible for _______. (explain)
    • constitutive 
    • β-galactosidase 
    • permease
    • pg 522-523
  41. A general rule derived from these experiments is that if a gene encodes a diffusible element (usually a protein) that can bind to target sites on any DNA molecule in the cell, whichever allele of the gene is _______ will override any other allele of that gene in the cell (the ______ allele therefore acts in trans). If a mutation is cis-acting, it affects only the expression of adjacent genes on the same DNA molecule; it does this by altering a DNA site, such as a _______-______ site, rather than by altering a ________-_______ gene
    • dominant 
    • dominant
    • protein-binding site
    • protein-encoding gene
  42. When scientists became able to purify the Lac repressor protein, they could verify that it can in fact bind _______ to operator DNA. The researchers mixed together __________ labeled repressor protein and a ________ _______ DNA that contained _____ _______. (How does it work?)
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    • physically
    • radioactively 
    • bacterial virus
    • lac operon 
    • pg 523 right
  43. The purified repressor protein is a dimer of two identical _____-encoded subunits; in some situations, two dimerss of Lac repressor can associate to form a ________. Importantly, each subunit contains _____ distinct domains. One of these regions binds to the ______, while a second domain _______ and binds to DNA at _______ sites
    Image Upload 16
    • lacI-encoded subunits
    • tetramer
    • three 
    • inducer
    • recognizes
    • operator
  44. When researchers investigated the molecular nature of mutations that affected repressor functions, their results made complete sense with this picture of the _______ subunits. lacI- mutations encoding proteins that could not bind to the operator affected amino acids in the protein's _____-______ domain. In contrast, lacIs superrepressor mutations, encoding proteins that could not be _______, were clustered in codons for amino acids in the ______-_____ domain
    • repressor subunits
    • DNA-binding domain 
    • induced
    • inducer-binding domain
  45. The third domain of a Lac repressor subunit, which is found at the _______ region of the polypeptide, interacts with the ______ _____ of other subunits to allow formation of the _____ and _______ proteins.
    • C-terminal 
    • same domain 
    • dimeric and tetrameric
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  46. X-ray crystallographic studies revealed that the DNA-binding domain of Lac repressor subunits has a characteristic three-dimensional structure: Two α-helical regions are separated by a turn. This _____ ____ ______ motif in the protein fits well into the ______ groove of the DNA
    • helix-turn-helix (HTH) motif 
    • major groove
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  47. In the 1970s, geneticists studying gene regulation developed new in vitro techniques to determine where regulatory proteins bind to the DNA. Purified proteins that bind to fragments of DNA protect the region to which they bind from ________ by enzymes such as DNase-I that break the ________ bonds between nucleotides
    • digestion
    • phosphodiester bonds
  48. If a sample of DNA, labeled at one end of one strand and bound by a purified protein, is digested partially with DNase-I, the enzyme will cleave any given _________ bond in at least some DNA molecules in the sample, except for those __________ bonds that are in regions protected by the ______ protein
    • phosphodiester bond
    • phosphodiester bonds 
    • bound
  49. Gel electrophoresis of the DNA and autoradiography (exposure of the gel to radiation-sensitive film) reveal bands at positions corresponding to the _______ between each base, except in the region where ______ protein protected the ____. Portions of the gel without bands are thus "footprints." What does this indicate?
    • cleavage 
    • bound 
    • DNA
    • indicating the nucleotides of the DNA fragment that were protected by the DNA-binding protein 
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  50. DNA footprinting experiments showed that some of the nucleotides in the lac operon operator are also part of the lac operon ________ (explain)
    • promoter
    • that is, the nucleotide sequence for the promoter, where RNA polymerase first binds to the gene and the sequence for operator overlap
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  51. In fact, some of the nucleotides in the operator are actually transcribed into mRNA when the operon is ______ _____. These observations mean that if the operator site is occupied by the _______ (as would happen in a normal cell in the absence of lactose), RNA polymerase cannot _____ the promoter nor _____ to it. These finding explain in a straightforward manner why binding of the repressor to the operator blocks expression of the ____ _____ genes
    • turned on 
    • repressor 
    • "see" 
    • bind 
    • lac operon
  52. Two dimers of the Lac repressor protein can associate to form a _______. DNA footprinting experiments performed with tetrameric Lac repressor, and with ______ fragments of lac operon DNA that include more sequences ______ of the promoter, revealed that the Lac repressor protein can actually bind to _______ sites in the vicinity of the ________; One of these sites is called o1 (o for _______; this is the site originally identified by oc mutations); the other sites are o2 and o3
    • tetramer
    • larger
    • upstream
    • three
    • operon 
    • operator 
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  53. Site o1 has the strongest binding affinity for the _______, and one of the dimers making up the tetramer always bind to the rotationally ________ sequences at this site. The other dimer within the tetramer binds to either o2 or o3. Mutations in either ____ or ____ thus have very little effect on repression. By contrast, mutations in both _____ and ____ make repression ____ times less effective. The conclusion is that for maximal repression, _____ dimers (and thus all four of the tetrameric repressor's subunits) must bind DNA __________
    • repressor
    • symmetrical
    • o2 or o3
    • o2 and o3
    • 50 
    • two 
    • simultaneously
  54. The o1 site is located roughly ____ bases away from o2 and _____ bases away from o3. These distances are sufficiently ______ so that a tetrameric repressor molecule can bind ________ to o1 and either o2 or o3 only if a _____ of DNA forms between operator sites
    • 400
    • 100
    • large
    • simultaneously
    • loop
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  55. Binding at ______ _________ _________ (two in each of two operator sites) increases the stability of the protein-DNA interactions so much that it can compensate for any _____ required to form the loop. In fact, the DNA binding of the Lac repressor is so _______ that only 10 repressor tetramers per cell are sufficient to maintain _______ in the absence of _______
    • four recognition sequences
    • energy
    • efficient 
    • repression 
    • lactose
  56. ______ is only one of the sugars bacterial cells can use as sources of energy and carbon atoms. In fact, if given a choice, most bacteria prefer ______. E. coli grown in medium containing both ______ and ______, for example will deplete the _____ before gearing up to utilize ______. While the ______ is present, the cells do not turn on expression of the ____ proteins even if _______ is present
    • Lactose 
    • glucose
    • glucose and lactose
    • glucose
    • lactose
    • glucose
    • Lac proteins
    • lactose
  57. Why doesn't lactose act as an inducer under these conditions?
    pg 526 mid right
  58. Inside bacterial cells, the small nucleotide known as _______ (cyclic adenosine monophosphate) binds to a protein called ______ _____ protein. The binding enables _____ to bind to DNA in the rgulatory region of the lac operon near the _________
    • cAMP
    • cAMP receptor protein (CRP)
    • CRP
    • regulatory region 
    • promoter
  59. When bound to DNA, CRP helps recruit _____ _______ to the promoter by making ______ contacts with the polymerase enzyme; in essence, then, the DNA binding of CRP ________ the ability of RNA polymerase to _______ the lac genes
    • RNA polymerase
    • physical
    • increases
    • transcribe
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  60. CRP thus functions as a _______ regulator that enhances the transcriptional activity of RNA polymerase at the lac promoter, while cAMP is an _______ whose binding to CRP enables CRP to bind to _____ near the _________ and carry out its _______ function
    • positive
    • effector
    • DNA
    • promoter
    • regulatory
  61. effector
    a small molecule that binds to an allosteric protein or an RNA molecule and causes a conformational change (an inducer such as allolactose is, like cAMP, an effector that activates gene expression)
Card Set
Gene Regulation in Prokaryotes
Ch 15.1~15.3