CHAPTER 16: Gene Regulation in Prokaryotes
riboswitches
allosteric RNA leaders that bind small molecule effectors to control gene expression.
a protein that can undergo reversible changes in conformation when bound to another molecules is called a(n)
allosteric protein
a pathway that is turned on only in the presence of cerytain molecules in the cell's environment is under _______ regulation
inducible
lacA operon encodes for
transacetylase
the entire set of RNAs produced by a cell is called its...
transciptome
riboswitches can regulate....
transcription or translation
prokaryotes regulate gene expression by activating, inhibiting, increasing or decreasing the _______ of specific gene, and.or interfering with the _______ of the mRNAs of those genes.
transcription;translation
exposure of E. coli cells to lactose increases levels of lactose-utilizing proteins
1,000-fold
what are the steps for the mechanism of quorum sensing...
1. Luxl synthesizes and releases autoinducer 2. autoinducer eneters the cell 3. autoinducer reaches a threshold 4. autoinducer binds LuxR 5. bioluminescence operon is transcribed
what are the steps for contrusting a directional cDNA library for RNA-seq.
1. bacterial RNAs are fragmented w, RNase 2. 5' ends are dephosphorylated enzymatically 3. RNA ligase is used to ligate a short synthetic single-stranded RNA sequence (adapter A) to the 3' ends 4. 5' ends are rephosphorylated enzymatically 5. RNA ligase is used to ligate a short synthetic with a different base sequence (adapter B) to the 5' ends of the RNA fragments 6. cDNAs are synthesized from these templates
the RNA leader of the trp operon includes a short open reading frame containing _____ codons, _____ of which code for tryptophan
14; 2
DNA footprinting experiments have revealed that the lac operon has a total of ____ operator sites
3
in the trp RNA leader, ________ stem-loop structures are possible
3
in _____-___________ transcription termination, a sequence of bases in the RNA forms a hairpin loop that serves as the signal for the dissociation of the RNA polymerase from the DNA and the release of the completed RNA
Rho-independent
aptamer
The region of a riboswitch that serves as a receptor for a ligand; binding of the ligand to the aptamer triggers conformational changes in the expression platform that influence gene expression.
a mutation in the operator that renders it nonfunctional will cause what form of expression for the lac operon
a constitutive expression
lac repressor is
a dimer or a tetramer
RNA-Seq
a method for analysis of the transcriptome of an organism in which millions of cDNAs are sequenced. Also called cDNA deep sequencing.
reporter gene
a protein-coding region of a gene incorporated into a recombinant DNA molecule along with putative DNA regulatory elements. After transformation of bacteria or incorporation of the reporter gene into the genome of an organism, the reporter gene "reports" the activity of the putative regulatory elements by expressing the protein.
operator site
a short DNA sequence near a promoter that can be recognized by a repressor protein; binding of repressor to the operator blocks transcription of the gene.
lac operon
a single DNA unit in E. coli, composed of the lacZ, lacY, and lacA genes together with the promoter (P) and operator site (o), that enables the simultaneous regulation of the three structural genes in response to environmental changes.
effector
a small molecule that binds to an allosteric protein or RNA and causes a conformational change.
stem loops (or hairpin loops)
a structure formed when a single strand of RNA folds back on itself due to complementary base pairing between different regions in the same molecule. Also called a hairpin loop.
the transport of glucose into a bacterial cell causes the intracellular level of cylic-AMP to fall because the eznyme ____ _____ is inhibited
adenyl cyclase
the binding of ________ to the lac repressor promotes a conformational change that prevents the repressor from binding to the lac operon
allolactose
RNA leader sequences are _______ in that they can alter their stem-loop structures and thus their fxn. in response to a wide variety of environmental cues
allosteric
the binding of a small effector molecule to a regulatory molecule alters the regulatory protein's conformation, and that..
alters the protein's ability to bind to DNA
RNA thermometer
an RNA leader that regulates translation in response to temperature through a stem-loop structure whose stability is temperature-dependent.
CRP (cAMP receptor protein) of the lac operon fxns as
an activator
the end product of an anabolic pathway can bind to the ____ of the riboswitch, this would shift the conformation of the leader so that it can now block the ribosome from binding
apatamer
by studying the lack of transcription of the trp operon in the absence of the Trp repressor, Charles Yonofsky discovered
attenuation
the trp operon is regulated by a repressor protein and by _____ in which transcription is stopped prematurely
attenuation
lacZ operon encodes for
beta-galactosidase
because it is easy to measure the levels of the ______-________, the lacZ gene which encodes this enzyme can be used as a(n) ____ gene to detect the amount of transcription that occurs in response to any specific regulatory element
beta-galactosidase; reporter
anabolic pathways
biochemical pathways for the synthesis of complex molecules from simpler ones.
beta-galactosidase
breaks down lactose into glucose and galactose
inducible operons usually encode ____ enzymes, and repressible operons usually encode ____ enzymes
catabolic; anabolic
induced heat-shock proteins include the so-called molecule _________ which help refold proteins that have been denatured by heat
chaperones
consider a bacterial w. the genotype: lacl+ o+ lacZ+ lacY-/F' ( lacl+ o+ lacZ- lacY+). the expression of the gene lacZ is ______ and the expression of the gene lacY+ is ______...
constitutive; inducible
in E. coli the sigma factor that mediates a globl heat shock response is sigma^32. it binds to the ____ RNA ________, thereby allowing the holoenzymes to recognize specific ______ and to initiate transcription of the genes encoding heat shock proteins
core; polymerase; promoters
mechanisms possible for antisense RNAs
degradation of mRNA, inhibition of mRNA transcription and translation
cis
describes the action of a DNA site or an RNA molecule that acts only on the DNA or RNA to which it is connected physically.
trans
describes the action of a protein or RNA that can bind to target sites on any DNA or RNA in the cell.
cAMP-CRP complex is a ____, and the lac operon site to which it binds contain rationally ______ DNA sequences. the complex contacts _____ _______ directly to help in transcription initiation
dimer; symmetric; RNA polymerase
the lacl+ allele is _____ to lacl- in ______
dominant; trans
pos. regulation and neg. regulation typically refer to mechanisms that...
enhance or inhibit RNA polymaerase activtiy, respectively
genes in the trp operon encode
enzymes used in tryptophan biosynthesis
T/F: a particular segment of mRNA from the trp operon can participate in the formation of more than one stemp loops at the same time
false
T/F: effector molecules influence gene expression by binding to DNA
false
T/F: the RNA leader of the trp operon encodes an enzyme
false
T/F: the lactose-utilization proteins are totally absent in cells grown w/o lactose
false
T/F: the rate of transcription initiation rarely influences overall gene expression
false
T/F: regulatory sRNA molecules are usually 10-50 nts long
false (they're typically 50-400nts long)
the trp RNA leader has four regions that can...
form stem loops
in order to study cis-acting regulatory regions, scientists can make a _____ gene which contains the regulatory region, followed immediately by the lacZ coding sequence
fusion
repressible regulation
gene control where transcription occurs only in the absence of a repressor.
inducible regulation
gene control where transcription occurs only in the presence of an inducer.
the synthesis of RNAs and proteins according to the instructions encoded in DNA is a process called
gene expression
an inducer is a small effector molecule that causes transcription to..
increase
the CRP-cAMP complex ____ transcription in several ____ operons, including the gal operon and ara operon
increases; catabolic
the binding of the _____ to the repressor causes an allosteric effects that abolishes the repressor's ability
inducer
lacY operon encodes for
lac permease
in the PaJaMo experiment, the researchers transferred the ___ into a bacterial cell devoid of the _______ in a medium containing no lacotse
lacl+ and lacZ+ alleles; lacl and lacZ proteins
the bassis of the PaJaMo experimen was the transfer of E. coli chromosomal DNA from a donor cell with the genotype _____ ______ to a recipient cell with the genotype lacl- lacZ-
lacl+ lacZ+
Jacob and Monod studied _______ metabolism in E. coli
lactose
when the tryptophan levele in the cell ____, the ribosomes pauses in region 1 of the trp mRNA leader, causing region 2 to bind to region 3 and transcritpion to continue
low
VF genes encode subunit of the enzyme luciferase
luxA and luxB
VF genes recycle luciferase substrates and cofactors
luxC, luxE and lux D
Vibrio fischeria genes that encode the qurom-sensing proteins
luxR and luxl
a strain of bacteria containing an F' plasmid is called a _______, or a partial diploid
merodiploid
catabolic pathways
metabolic pathways by which complex molecules are broken down.
cDNA deep sequencing
method for analysis of the transcriptome of an organism in which millions of cDNAs are sequenced. Also called RNA-Seq.
the inhibition of RNA polymerase activity is termed ______ regulation, whereas the enhancement of RNA polymerase activity is known as _______ regulation
negative; positive
features of a superrepressor mutation that affects the lac operon
occurs in lacl causes constant repression of the lac operon prevents the repressor from binding to the inducer
the lac operon control region can help produce protein drugs in bacteria. the first step in this process is the creation of a recombinant plasmid in which the lac regulatory region is fused to the _______ _______ ______ encoding the protein to be expressed
open reading frame
a mutation that changes the nucleotide sequence of the ___, so that the repressor is no longer able to bind to the site, will cause a constitutive synthesis of the lactose-utilizing proteins
operator
if several genes or operons were regulated by the same repressor proteins, you would expect to find similar or related ______ DNA sequences just upstream to those genes or operons
operator
a loss-of-function mutation in a gene encoding a repressor protein has the same effect as a mutation in the
operator site
because there is an overlap between the sequences of the lac _____ and lac _____, binding of the repressor to the DNA will inhibit RNA piolymerase from binding. as a result the expression of the lac operon genes are blocked
operator; promoter
a(n) _______ is a group of two or more genes under the transcriptional control of a single promoter
operon
a operon encodes a _____ mRNA, an RNA that contains the sequence of two or more genes
polycistronic
to identify operons, computer experts have developed algorithms that seartch for several closely spaced genes adjacent to a(n) __________, as well as for transcription termination signals
promoter
transcription in prokaryoes begins when the RNA pol holoenzyme binds to specific DNA sequences in a region called the...
promoter
in the lac operon, the two main regulatory DNA elements are the ______ and the ______
promoter; operator
mutations in trans-acting element typically occur in ______, while mutations in cis-acting elements occur in______....
protein-encoding genes; protein-binding sites
DNase I footpringting technique is used to show where _____ bind to _______
protein; DNA
antisense RNAs
regulatory RNAs that are complementary in sequence to the mRNAs they regulate because they are transcribed using the opposite strand of DNA as a template. Antisense RNAs can block transcription or translation of their target mRNAs.
in prokaryotic transcription, the switch from initiation to elongation involves...
release of sigma factor
a pathway thaty is turned off when the cell has enough of the end product is under _____ regulation
repressible
two mechanism that control expression of the trp operon in E. coli
repression and attenuation
to initiate translation, ribosomes attach to the mRNA at a site called the _______-______ site
ribosome binding
an RNA leader whose secondary structure, and thus its effect on gene expression, is determined by whether or not it's bound to a small molecule effector is called a(n)....
riboswitch
when guanines leveles are high, guanine binds to the aptamer of it ________. in response, the expression platform forms a ______ structure, which shuts down the transcription of genes that participate in guanine synthesis
riboswitch; terminator
promoters for the production of ____ mRNAs and ________ regulatory RNAs are located on opposite sides of the coding region
sense; antisense
the lacl gene, which encodes the Lac repressor is...
separate from the lac operon and is unregulated
terminator
sequences in the RNA transcripts that cause RNA polymerase to stop transcription; stem-loop structures in an RNA leader sequence that stop transcription of the RNA downstream.
a total of _______ genes are used to control bioluminescene in VF
seven
genes induced by heat shock are recognized by an alternative sigma factor called...
sigma^32
RNA leader sequences can form secondary structures called ___ ________, which can terminate transcritpion of the rest of the mRNA permaturely
stem loops
constitutive mutants
strains that synthesize certain enzymes all the time, irrespective of environmental conditions.
the _______ mutations in the lacl gene render the protein product incapable of binding the inducer
superrepressor
in the trp RNA leader sequnce, the 3-4 stem-loop configuration is called a ______ because it leads to the production of a short, "attenuated" RNA
terminator
the lac repressor binds as a
tetramer: a dimer at o1 and a dimer at either o2 or o3
the historic PaJaMo experiment was used to demonstrate
that the lacl gene encodes a repressor protein
the CRP protein associates w. cAMP and binds to...
the DNA as a dimer
how does an effector molecule influence gene expression
the effector molecule binds to a regulatory protein and affects its ability to bind DNA
why is transcription of the lac operon low when both lactose and glucose are present.?
the low levels of cAMP result in CRP not binding to the CRP site
operon theory
the model that explains the repression and induction of genes in E. coli.
induction
the process by which a signal causes expression of a gene or set of genes.
the effector for a repressible operon may be...
the product of the metabolic pathway carried out by enzyme encoded by the operon
regulatory elements of the lac operon are...
the promoter and operator
radioactively-labeled repressor protein from lacl+ cells is mixed with a bacterial virus DNA that contains the wild-type lac operaot. the mixture is then centrifuged in a glycerol gradient. what is the likely result
the protein will cosediment w. the DNA
becateria regulate gene expression at the first step by inflencing...
the rate of transcription initiation
catabolite repression
the repression of transcription in sugar-metabolizing operons like the lac operon when glucose or another preferred catabolite is present.
when bacterial cells are grown in a medium w/o lactose,...
there are very low levels of the proteins required for lactose utilization
how are the two sigma factors sigma^70 and sigm^32 in E. coli different from each other?
they bind to different promter sequences and they denature at different temperatures
characteristics of antisense RNAs
they can be complementary to the entire mRNA or be complementary to only part of the mRNA
DNA footprinting experiment have revealed that the lac operon has a total of ___ operator sites
three
a protein that can diffuse through the cytoplasm and act at DNA target sites elsewhere in the genome is called a ____-acting element
trans
expression platform of the guanine riboswitch fxns. by controlling..
transcritption
sRNAs regulate...
translation in trans by base pairing with mRNAs
:ac permease fxn.
transports lactose into the bacterial cell
T/F: in the lac operonm site o1 has the strongest binding affinity for the repressor
true
the historic PaJaMo experiment was named after what scientists
Arthur Pardee, Jacob and Monod
repressors can be IDed by the presence of sequnces that encode
DNA-binding motifs such as the helix-turn-helix or zinc finger motifs
the helix-turn-helix motif is commonly found in
DNA-binding proteins
Monod and Jacob stidued lactose metabolism in the bacterial species
Escherichia coli
during the initiation step of transcription, DNA is unwound by the action of the enzyme...
RNA polymerase
positive regulators of transcription usually act be establishing a physical contact w ______ ________ which attracts it to the promoter or keeps it there longer
RNA polymerase
transcription is the work of
RNA polymerase
some bacterial mRNAs containe dleader sequences that regulate translation in response to termperature. this specialized mechanism, which can be regarded as a rudimentary riboswitch, is called a(n)
RNA thermometer
expression platform
The region of a riboswitch that changes conformation in response to a ligand molecule binding to the aptamer.
two main regions of a riboswitche...
aptamer and expression platform
how does tryptophan fxn. in the regulation of the trp operon?
it binds to the repressor and only then can this complex bind to DNA thus preventing transcription of the operon
beta-galatosidase activity can be dected using _______________ because beta-galactosidase cleaves it to a product w. a yellow color
o-nitrophenyl-galactoside (ONPG)
allosteric protein
proteins that undergo reversible changes in conformation when bound to another molecule (an effector).
inducer
a small molecule that causes transcription from a gene or set of genes.
antiterminator
a stem-loop structure in an RNA that prevents formation of a terminator.
DNase I footprinting
a technqiue used to study protein-DNA interactions
attenuation
a type of gene regulation in which transcription of a gene terminates in the RNA leader sequence before a complete mRNA transcript is made; involves interaction between stem loops in the RNA leader and the translation machinery.
effects of either a loss-of-function mutation in the lacl gene or a mutation in operator that prevents repressor binding have...
both result in constitutive expression of the lac operon
in what way can a riboswitch directly regulate translation
by blocking the ribosome binding site
how do regulatory sRNAs fxn. at the translational lelvel
by inhibiting or activating translation
postivie control of the lac operon involves a small nucleotide known as _____ which binds to a protein called _______. the binding of the nucleotide enables the protein to bind to DNA in the regulatory region of the lac operon. this binding increases the ability of ______ __________ to transcribe lac genes
cAMP; CRP (cAMP receptor protein); RNA polymerase
regulations of the lac and trp operons can be summarized as follows. in the presence of the inducer, the lac repressor ____ bind to the lac operatorp; and in the presence of tryptophan the TrpR repressor _______ bind to the trp operator
cannot; can
the somewhat imprecise term that describes how transcriptional regulation is influenced by glucose is...
catabolite repression
a DNA segment that must be adjacent to the gene(s) it regulates is called a ______-acting element
cis
the binding of a small effector molecule to a regulatory protein changes the _____ of the regulatory protein, which alters its DNA-binding ability
conformation
prokaryotic gene regulation
control of gene expression in a bacterial cell via mechanisms to increase or decrease the transcription or translation of specific genes or groups of genes.
mechanisms that are possible for regulatory sRNAs
inhibiton or activation of mRNA translation and degradtion of mRNA
lac operon structural genes are transcribed as...
part of a single polycistronic mRNA with three independent ribosome binding sites
attenuator
part of the RNA leader of some bacterial genes that forms alternate stem-loop structures that depend on interaction with the translation machinery. In one conformation, a stem loop (the terminator) terminates transcription; in the alternate conformation, a different stem loop (the antiterminator) forms and transcription continues.
compared to cells with a wild-type lac operon, mutants with a lac^s genotype show a(n)....
repressed transcriptional rate, independent of the presence or absence of lactose
consider a bacterial cell w. the genotype lacl+ o+ lacZ+ lacY+ that was transformed w. an F' plasmid carrying lacl^s o+ lacZ- lacY-. the expression of the genes lacZ+ and lacY+ in the merodiploid bacterium is....
repressible in the absence or presence of lactose
when tryptophan is absent from the growth medium, the Trp ______ protein cannot bind to the ________. as a result, the tryptophan biosynthetic genes are expressed
repressor; operator
in attenuation, the level of an a.a governs the conformation of the leader indirectly. However, leaders that act as _____ change conformation by binding a particular effector directly
riboswitches
sRNAs
small RNA molecules that regulate translation in trans by base pairing with sites on mRNAs that can hide or expose the ribosome binding site.
what determines the DNA binding specificity of a protein w. a helix-turn-helix motif
the sequence of a.a.s in the helices
a strain of E. coli that has a mutation in the lacl gene such that lac operon is expressed even in the absence of lactose is an _______ mutant
constitutive
mutations in the trpR gene that alter the protein';s DNA-binding domain will result in...
constitutive activation of the trp genes
bacteria can adjust their behavior according to their population density by using a communication system called
quorum sensing
repressor
a type of transcription factor that can bind to specific cis-acting elements and thereby diminish or prevent transcription. Repressors bind operators in prokaryotes and enhancers (or silencers) in eukaryotes.
operon
a unit of DNA composed of two or more genes transcribed as a polycistronic mRNA under the control of a single promoter and operator.