Gene Regulation in Prokaryotes II

  1. The CRP protein associated with cAMP binds to DNA as a dimer. Just as is the case with the _____ _____, the DNA sequece to which the CRP-cAMP complex binds has ______ ______. Thus, CRP-binding sites consist of _____ recognition sequences pointing in _______ directions, each able to bind one subunit of the _____ dimer
    • lac operator
    • rational symmetry
    • two 
    • opposite
    • CRP
    • *This example again stresses the importance both of the multimerization of DNA-binding protein subunits and of the clustering of their corresponding binding sites in the vicinity of promoters
  2. Glucose controls the amount of cAMP in the cell ______ (how). Thus, when glucose is present, the level of cAMP remain _____; when glucose is absent, cAMP synthesis ______. As a result, when glucose is present in the culture medium, there is ______ cAMP available to bind to ______ and therefore little _______ of the lac operon, even if _______ is present in the culture medium
    • indirectly
    • by decreasing the activity of adenly cyclase, the enzyme that converts ATP into cAMP
    • low 
    • increases
    • little 
    • CRP
    • induction 
    • lactose
    • Image Upload 1
  3. The overall effect of glucose in preventing lac gene transcription is known as _______ ______ (why?)
    • catabloic repression 
    • because the presence of a preferred catabolite (glucose) represses transcription of the operon
  4. You can see that having operons rely on the activity of two different regulators (_____ _____ and _____ in the case of the lac operon) that respond to different environmental cues __________
    • Lac repressor and CRP 
    • increases
    • *E. coli cells can adjust their gene expression exquisitely to benefit from the particular mix of sugar carbon sources or other nutrients available to them at any given time
  5. Although positive and negative regulators obviously have opposite effects on transcription, you should keep in mind taht most regulators of both types work through their effects on ____ ______. Name one way negative regulators, such as the Lac repressor, prevent initiation. Name one way how positive regulators get it done
    • RNA polymerase 
    • Negative regulators: by blcoking the functional binding of RNA polymerase to the promoter.
    • Positive regulators: by establishing a physical contact with RNA polymerase that attracts RNA polymerase to the promoter or that keeps RNA polymerase bound to the DNA longer so that it initiates transcription more frequently
  6. In bacteria, the multiple genes of both ______ and _______ pathways are often clustered together and _______ in operons. We have seen that catabolic lac operon responds to the presence of lactose by inducing _______ of the lac genes. By contrast, anabolic operons respond to the presence of the pathway's end product by ______ _____ expression of the structural genes whose protein products manufacture the end product. In other words, anabolic pathways require ________ regulation
    • catabolic and anabolic pathways
    • coregulated
    • expression 
    • shutting down 
    • repressible
  7. Several anabolic bacterial operons are involved in the production of _____ _____. A well-studied example is the E. coli ________ operon, a group of five genes (trpE, trpD, trpC, trpB, and trpA) required for biosynthesis of amino acid ________. Maximal expression of the trp genes occurs when tryptophan is _______ in the growth medium
    • amino acids
    • tryptophan (trp) operon
    • tryptophan 
    • absent
    • Image Upload 2
    • Image Upload 3
  8. The trp operon is controlled in part at the level of transcription initiation through the action of a ________ protein that is the product of the ______ gene. In contrast to allolactose, which functions as an ______ that prevents the Lac repressor from binding to the _______, tryptophan functinos as a _______ for the TrpR repressor protein
    • repressor 
    • trpR gene
    • inducer
    • operator
    • corepressor
  9. That is, tryptophan is a small-molecule _______ whose binding to the TrpR protein allows this _____ regulator to bind to DNA and inhibit ________ of the genes in the trp operon
    • effector 
    • negative
    • transcription
  10. The binding of tryptophan to the TrpR repressor causes an _______ alteration in the _______'s shape, and only with this _______ can the TrpR protein bind to the operator site. Mutation in the trpR gene that change either the protein's _______-_______ domain or its _____-______ domain _______ the TrpR repressor's ability  to associate with DNA, and both types of mutations therefore result in the ______ _______ of the trp genes even when tryptophan is present in the growth medium
    • allosteric 
    • repressor's shape
    • alteration 
    • tryptophan-binding domain 
    • DNA-binding domain 
    • destroy
    • constitutive expression 
    • read pg 528 right mid
  11. Many bacterial genes, lie those in the lac and trp operons, are regulated primarily at the level of transcription initiation by the action of _______-acting regulatory proteins such as repressors or CRP that bind to ____-acting DNA sites like ______ or ____-binding sites. Allostery of the regulatory proteins (explain) enables genes to _____ ____ or ____ ____ in response to the cellular environment
    • trans-acting 
    • cis-acting 
    • operators or CRP-binding sites
    • that is, changes in their shapes that occur when they bind to small molecules like allolactose or tryptophan 
    • turned on or turned off
  12. All bacterial mRNAs begin with an untranslated region called the 5' UTR, or ______ _____ sequence. Through complementary base-pairing, many RNA leaders form secondary structures called _____ ______ (or _____ _____). The _____ _____ can terminate transcriptionof the rest of the mRNA prematurely, or they can prevent translation by blocking access of the mRNA to the _____ _____ site. These RNA leaders are _______ in that they can alter their stem-loop structures and thus their ______ in response to a wide variety of environmental cues
    • RNA leader sequence 
    • stem loops (or hairpin loops) 
    • stem loops
    • ribosome binding site 
    • allosteric
    • function
  13. The first RNA leader mechanism discovered involves fine-tuning of the response of the trp operon to the amount of tryptophan available to the cell. Keep in mind that maximal expression of trp genes occurs when tryptophan is absent from the growth medium: (explain)
    • The TrpR repressor protein cannot bind to the operator because the corepressor tryptophan is lacking 
    • *The TrpR-mediated repression of the trp operon is indeed a critical first step, but it is not the only regulatory mechanism controlling expression of the trp genes in E. coli
  14. If TrpR binding to the repressor were the only event of importance, you would expect trpR- mutants to show ______ ______ of their trp genes. With or without tyrptophan in the medium, if there is no repressor to bind at the operator, RNA polymerase would have uninterrupted access to the _____ ______.
    • constitutive expression 
    • trp promoter
  15. Surprisingly, the actual experiments showed that the trp genes of trpR- mutants are not completely _________ when tryptophan is present in the growth medium. The removal of tryptophan from a medium in which trpR- mutants are grwoing cuased expression of the trp gene to increase _______. Apparently, tryptophan can affect the expression of the trp operon by some kind of additional mechanism that does not involve the _____ _______ protein
    • de-repressed (that is, turned on)
    • threefold
    • TrpR repressor protein
  16. In a series of elegant experiments, Charles Yanofsky and coworkers found that this repressor-independent change in trp operon expression involves the production of alternative ____ _____ ______. Sometimes initiation at the promoter leads to transcription of a _______ mRNA ~ 140 bases long containing only the _____ _____ but none of the structural genes
    • trp operon transcripts
    • truncated
    • RNA leader
  17. At other times, transcription continues beyond the end of the _____ sequence to produce a _____ operon-length transcript. In analyzing why some mRNAs terminate before they can transcribe the structural trp genes, while others do not, the researchers discovered ________ (define). Whether or not transcription terminates depends on how the translation machinery reads the ______ structure of the _____ _____
    Image Upload 4
    • leader
    • full
    • attenuation: control of gene expression by RNA leader-mediated premature termination of transcription 
    • secondary 
    • RNA leader
  18. The trp RNA leader can fold into _____ different stable conformations, each one based on the complementarity of bases in the same molecule of RNA. The first conformation contains two ____ ____ structures: Reegion 1 makes a ____ _____ with region 2, while Region 3 ______ with region 4. The 3-4 stem-loop configuration is called a _______ (why?)
    Image Upload 5
    • stem loop structures
    • stem loop
    • associates
    • terminator
    • because when it forms in the trp operon transcript, RNA polymerase contacts it and stops transcription, producing a short, "attenuated" RNA
  19. The alternative RNA structure, called the _______, forms by ______ ______ between regions 2 and 3. In this confomration, the leader RNA cannot form the ________ (why and what is the result?)
    • antiterminator
    • base pairing
    • terminator
    • because region 3 is no longer available to pair with region 4 
    • result: the transcription machinery continues to produce a full-length mRNA that includes the trp structural gene sequences
  20. The early translation of a short portion of the ____ _____ (while transcription of the rest of the leader is still taking place) determines which of the two alternative RNA structure forms. That key portion of the RNA leader includes a short _____ ____ ______ containing 14 codons, two of which are _____ codons. When tryptophan is present, the ribosome moves quickly past the trp codons in the _____ _____ and proceeds to the end of the ______'s codons allowing formation of the _______
    Image Upload 6
    • RNA leader 
    • open reading frame 
    • trp 
    • RNA leader
    • leader's 
    • terminator
    • Image Upload 7
  21. In the absence of tryptophan, the ribosome stalls at the two trp codons in the RNA leader because of the lack of ______ ______ in the cell. The ________ is then able to form, which prevents formation of the terminator. As a result, transcription proceeds through the ______ into the structural genes
    • charged tRNATrp
    • antiterminator
    • leader 
    • Image Upload 8
  22. It has become increasingly clear that RNA-based mechanisms do much more than fine-tune the expression of certain ______. Shifts between different conformation of RNA molecules play the primary role in the regulations of many genes. One wide-spread mechanism involves ________ (define)
    • operons
    • riboswitches: allosteric RNA leaders that bind small molecule effectors to control gene expression 
    • Image Upload 9
  23. Whereas in attentuation the level of an amino acid like tryptophan governs the ________ of the leader indirectly (through participation of ______ and _______), leaders that act as riboswitches have a region called the _______ that binds a particular effector directly. Riboswitches also have a second region, called the ______ _____, which controls gene expression by altering its _____ _____ structures in response to the _______ configuration
    • conformatino 
    • tRNAs and ribosomes
    • aptamer
    • directly
    • expression platform
    • stem-loop
    • aptamer
  24. In some riboswitches, the expression platform controls the termination of transcription. For example, one of the simplest riboswitches control transcription in response to guanine (3-story)
    Image Upload 10
    pg 530-531
  25. In other riboswitches, the expression platform regulates translation by ______ or ______ the ribosome binding site. In an anabolic pathway, binding of the ______ (in this case the end product) to the ______ would shift the conformation of the _____ so as to block the _______ ______ site, conserving cellular energy by preventing the synthesis of _______ products that are not currently needed by the cell
    Image Upload 11
    • blocking or unblocking
    • effector
    • aptamer
    • leader
    • ribosome binding site
    • protein
  26. Bacterial genomes encode many _____ ____ molecules (_____), that regulate translation in the ______ by base pairing with ______.  Regulatory ______ are typically 50-400 nt long, and their sequences contain a region complementary to several different mRNA targets. Most are sRNAs are ________ (explain)
    • small RNA molecules
    • SRNAs
    • trans
    • mRNAs
    • sRNAs
    • repressive
    • meaning that they inhibit translation of their target mRNAs by base pairing with the ribosome binding site 
    • Image Upload 12
  27. Some sRNAs, however, activate translatino of their target mRNAs by disrupting the formation of a _____ _____ structure in the ______ of the mRNA that would otherwise block the ______ ______ site. Name another way in which certain sRNAs scan influence the expression of particular target genes. 
    * = not likely tested
    Image Upload 13
    • stem-loop
    • leader
    • ribosome binding site
    • by promoting the degradation of the mRNA 
    • *-the double-stranded RNA region resulting from sRNA binding to the mRNA causes the mRNA to be degraded by ribonuclease enzymes
  28. You should note that most sRNAs do not bind to _______, so the sRNA usually cannot respond to environmental changes ______. Instead, the _______ or ______ of an sRNA is often controlled through other regulatory molecules, suchas transcription factors that interact with the sRNA gene promoter so as to _____ or ______ the cellular concentration of the sRNA (explain)
    • effectors
    • directly
    • transcription or stability
    • increase or decrease
    • SRNAs often act as intermediaries in regulatory cascades in which one regulator influences the expression of a different regulator
  29. The regulatory sRNAs just described are encoded by genes that can be far removed from the genes encoding their mRNA targets. In contrast, some bacterial genes are regulated by RNAs that are ________ in sequence to the mRNA because their transcription template is the _______ strand of DNA. These regulatory RNAs are called _______ ____; the mRNAs they regulate are ______ ______.
    • complementary 
    • opposite
    • antisense RNAs
    • sense RNAs
    • Image Upload 14
  30. Antisense RNAs range in size from 10-100 nt, and may be complementary to the entire ____ encoded on the ______ DNA strand, or they may ______ only part of it
    • mRNA 
    • opposite
    • overlap 
    • *Note that the promoters for the production of sense mRNAs and antisense regulatory RNAs are located on opposite sides of the coding 
    • Image Upload 15
  31. Some antisense RNAs function like the trans-acting sRNAs: They ______ translation by base pairing with the sense mRNA and ______ the ribosome binding site. In other cases the ______-stranded RNA formed by base pairing between the ____ ____ and _____ _____ can be degraded by ribonucleases
    Image Upload 16
    • inhibit 
    • blocking
    • double-stranded
    • sense mRNA and antisense RNA
  32. In yet other cases, it is not the antisense RNA itself, but the at of _______ it that inhibits expression of the ____ gene; that is antisense ________ can interfere with initiation of ________ of the sense gene
    • transcribing 
    • sense
    • transcription 
    • transcription
  33. One of the key advantages of the lac operon is the existence of simple assays to measure the amount of β-galactosidase enzyme in a sample (explain)
    Enzyme function can turn one colorless substrate into a soluble yellow compound, while β-galactosidase can also change the colorless compound X-gal into an insoluble blue precipitate
  34. Why can the lacZ gene that encode β-galactosidase serve as a reporter gene to detect the amount of transcription that occurs in response to any specific regulatory element?
    because it is so easy to measure β-galactosidase
  35. For this purpose, researchers use _________ DNA methods to create DNA molecules in which the coding region of lacZ is fused to ____-acting regulatory regions (including promoters and operators) of any other gene (gene X)
    • recombinant DNA
    • cis-acting
  36. The ______ ______ gene can then be introduced by transformation into bacterial cells. In cells with this ______ gene, conditions that normally induce the expression of gene X will generate _________. The scientists can then assess the activity of the gene X regulatory elements by monitoring the amount of _________ the cell produces
    Image Upload 17
    • synthetic reporter
    • fusion gene
    • β-galactosidase
    • β-galactosidase
  37. The availability of _____ _____ genes making it possible to identify the DNA sites necessary for ________ as well as the _____ and _______ involved in that regulation. State two examples
    • lacZ reporter genes
    • regulation
    • genes and signals
    • pg 533 left bottom
  38. Reporter fusions also make it possible to identify many of the genes that are regulated by the same _______ _______. To this end, researchers can use _______ to insert the lacZ gene, without its _______ region, at various sites around the bacterial chromosome. The lacZ gene cannot by itself make β-galactosidase, because it lacks a ________
    • environmental stimulus
    • transposons
    • regulatory 
    • promoter
    • Image Upload 18
  39. However, in some cells, the promoterless lacZ reporter gene becomes inserted _______ to the promoters and regulatory regions of other genes. If conditions are right to turn on the expression of a gene into which the reporter has inserted, the cell will produce _________. Using this method, researchers identified a set of genes activated by exposure to DNA-damaging agents like ____ light
    • adjacent
    • β-galactosidase
    • UV light
    • Image Upload 19
  40. When constructing a reporter gene, the crucial part of the lac operon is the _____ coding sequence. However, geneticists can exploit other parts of the lac operon, namely the regulatory region that allows high levels of transcriptiono, for a very important practical application (results?)
    What is the basic idea (4-story)
    Image Upload 20
    • lacZ
    • pg 534 upper left
    • pg 534 right below
  41. Transcriptome sequencing (define transcriptome) requires the production of a _____ library and subsequent sequencing of many of these ______; the entire procedure is called ____-____ or sometimes _____ ____ sequencing
    • transcriptome: is the base sequence of every transcript that a cell produces under a particular set of conditions.
    • cDNA library
    • cDNAs
    • RNA-Seq
    • cDNA deep sequencing
  42. Current RNA-Seq technology allows a researcher to obtain about one billion sequence "reads" of _____ sequence, each read about 150 nt long, in a single experiment. The higher the level of a particular mRNA in a cell, the ______ times cDNAs corresponding to that will be _______. Deep sequencing thus enables a researcher to quantify the levels of _______ of individual mRNAs as the number of cDNA sequence reads per ____ _____ of the genome
    • cDNA
    • more
    • sequenced
    • expression
    • base pair
  43. By comparing the transcriptomes of population of bacterial cells grown in different environments, investigators can determine which of all of the genes in the genome have their expression by what two means?
    pg 534 right mid
  44. In contrast with mRNAs in euks cell, _____ _____ are not characteristic features of mRNAs in prok organisms. As a result, cDNA libraries for bacterial cells must be constructed in a ______ manner from those in euk cells, which depend on the existence of _______ _____. In place of the ____ _____, investigators use ______ ______ to connect fragments of bacterial RNAs with _______ ________
    • poly-A tails
    • different 
    • poly-A tails
    • poly-A tails
    • DNA ligase
    • adapter oligonucleotides
    • Image Upload 21
  45. Why is it that the cDNA library must be constructed in a way that indicates which strand of the cDNA was copied first? What is the result?
    • because some genes are transcribed in both sense and antisense directions
    • this would track which cDNA strand sequence corresponds to the RNA and which one is the reverse complement
  46. directional cDNA library
    a cDNA library constructed so as to indicate the 5' ends and 3' ends of the original template mRNAs
  47. To analyze the data from an RNA-Seq experiment, a computer lines up each _____ sequence read with the ______ sequence of the bacterium. Explain the data for one small segment of a bacterial genome sequence example (3-story)
    Image Upload 22
    • cDNA
    • genome 
    • pg 535 top right
  48. One important example of the RNA-Seq method is found in studies of the ______ _____ response in E. coli resulting from exposure of the cells to extremely high temperatures (45 °C). RNA-Seq has found that high temps specifically induce the transcription of a suite of genes encoding specialized _____-_____ proteins _______ the tendency of high temps to cause the _______ and _______ of most of the other proteins in the cell. Some of the induced heat-shock proteins _______ and _______ aberrant proteins, while other heat shock proteins act as so called ________ _________ which help refold other proteins and also prevent their ________
    • counteract
    • denaturation and aggregation 
    • recognize and degrade
    • molecular chaperones
    • refold
    • aggregation
  49. E. coli's induction of the proteins that combat heat shock is a highly ______ stress response. Organism as different as bacteria, flies, and plants induce similar proteins in response to _____ temps
    • conserved
    • high
  50. New _____-acting regulatory proteins can be identified by searching the genome for sequences that could encode known DNA binding motifs, such as the _____ _____ _____ region of the Lac repressor protein. Computers can in some cases find the ____-acting sites bound by the _____-acting regulatory proteins by looking at the DNA sequences of the suites of genes that exhibit similar _______ in response to a given environmental condition
    • trans-acting  
    • helix-turn-helix region
    • cis-acting
    • trans-acting
    • regulation
  51. High temps induce transcription of the _____-______ genes, whose products protect cells from the _______ consequences of this environmental condition. Examination of the DNA sequences just ______ of the heat-shock gene coding region showed that the ______ of the heat shock genes have significant differences from the ______ of the everyday "housekeeping" genes expressed at ______ temp. (why?)
    Image Upload 23
    • heat-shock 
    • deleterious
    • upstream
    • promoters
    • promoters
    • normal 
    • The reason turns out to be that alternative sigma factors allow RNA polymerase to recognize the two different classes of promoters
  52. The normal housekeeping sigma factor, σ70, is active in the cell under nomral physiological conditions, but it becomes _______ and therefore is inactivated at ______ temps. By contrast, the alternative σ32 can function at _____ temps; it also recognizes different ________ sequences than those recognized by σ70
    • denatured
    • high 
    • high 
    • promoter
  53. Many genes induced by heat shock contain ______ _______ in their _______ that are recognized by σ32. How does σ32 factor mediates the heat-shock response?
    • nucleotide sequence
    • promoters
    • by binding to the core RNA polymerase, thereby allowing the polymerase to initiate transcription of the genes encoding the heat shock proteins
  54. Regulatory RNAs can be difficult to find. Although RNA leader devices such as ______ often have similar ________ structure features, little or no base sequence ______ exists that computers could identify from the genome sequence alone
    • riboswitches
    • secondary structure
    • conservation
  55. However, judicious examination of the transcriptome can supply useful clues for the discovery of ____ ______ devices, ______, and ______ ______. ______ ______ would be located at the 5' ends of transcripts connected to the _____ ______ ______ of genes. sRNAs should have ______ regions displaying at least limited _________ to portions of mRNA transcript
    Image Upload 24
    • RNA leader devices
    • sRNAs
    • antisense RNAs
    • RNA leaders
    • open reading frames
    • short
    • complementarity
  56. These regions of complementarity should be evenmore pronounced in the case of antisense RNAs, and the ______ _______ of the sense and antisense transcripts should be revealed by _________ of the corresponding cDNAs
    • inverse polarites 
    • directionalities
  57. Computerized analysis of the mRNA encoding the alterinative sigma factor σ32 revealed the existence of a specialized kind of RNA leader mechanism called an _____ ______, which can be regarded as a rudimentary ________. Such _____ ________ inhibit translation at _____ temps by forming _____-_____ structures that involve the ______ binding site in base pairing interactions, preventing mRNA _________
    • RNA thermometer 
    • riboswitch
    • RNA thermometers
    • low 
    • stem-loop 
    • ribosome 
    • translation
  58. At higher temps, the stem-loop structure becomes ______ and ______, freeing the _______ binding site. Why does this mechanism make perfect sense for the σ32 mRNA?
    Image Upload 25
    • unstable and unzips
    • ribosome
    • pg 537 left mid
  59. Quorom sensing
    Quorum sensing— universal communication mechanism used in bacteria to regulate gene expression in response to population density

    *fischeri are bioluminescent bacteria

    Fluoresce when population of bacteria is sufficiently large to produce a certain amount of light

    Helps squid (and bacteria) avoid predators at night
  60. The researchers constructed a V. fischeri ______ library in plasmids and used it to transform E. coli. Remarkably, a few E. coli colonies became ________. Even more remarkably, these light-producing bacteria began to glow only when the culture reached a high _____ of cells. Therefore, the recombinant plasmid in the E. coli cells contained all the V. fischeri ________ genes, and all the _______ elements needed for ______ _______. This plasmid contained only 9kb of V. fischeri DNA
    • genomic 
    • bioluminescent
    • density
    • bioluminescence 
    • regulatory
    • quorum sensing
  61. To determine how many bioluminescence genes were contained in the cloned 9kb V. fischeri genomic DNA fragment, the researchers grew bioluminescent E. coli strain in the presence of a ______ ______ and screened the colonies for ______ that could no longer produce _______
    • chemical mutagen 
    • mutants
    • light
  62. The scientists then sorted the mutations in the plasmid's V. fischeri genes into ________ groups. Colonies did not produce light if the two plasmids failed to complement (why?)
    • complementation groups
    • because they had mutations in the same V. fischeri gene
  63. Out of the two lux promoters, how many genes were transcribe? What was the breakdown of genes per promoter?
    Seven genes transcribed from two promoters

    One promoter produces luxR

    • The other results in transcription of luxI, luxC, luxD, luxA, luxB, and luxE
    • Image Upload 26
  64. The 9kb genomic DNA fragment also contains the two genes that mediate quorom sensing in V. fischeri: _____ and _____. LuxR protein is a type of transcriptional ______ protein called a ______, and it is needed for transcription of luxICDABE mRNA
    • luxI and luxR
    • activator
    • receptor 
    • Image Upload 27
  65. LuxI protein is a _______ enzyme which generates a molecule called an ________ which is an _______ that binds to _____ and enables the ______ to bind DNA
    • synthase 
    • autoinducer
    • effector
    • LuxR 
    • receptor
  66. The ________ is released into the extracellular environment, and it can also _____ the cell. When the bacterial population is ______, the cell cytoplasm is filled with ________, which binds _____; the bioluminiescence genes are expressed at _____ levels and the bacteria produce ______
    • autoinducer
    • reenter
    • dense
    • autoinducer
    • LuxR
    • high 
    • light
    • Image Upload 28
  67. When only a few bacteria are present, the _______ concentration is too low for much of it to have ______ the cell, the bioluminescence genes are transcribed only at _____ levels, and no _____ is produced
    • autoinducer
    • reentered
    • low 
    • light
    • Image Upload 29
Card Set
Gene Regulation in Prokaryotes II