Chapter 7 Chapter 7
1.
1. DesDescricribe the mobe the molecleculaular evenr events thats that occur at thet occur at the laclac operon whenoperon when E. coliE. coli cells arecells are shifted from a glucose-containing medium to
shifted from a glucose-containing medium to a lactose-containing medium.a lactose-containing medium. Answer:
Answer:
When no lactose is present, binding of the
When no lactose is present, binding of the laclac repressor to a seuence called therepressor to a seuence called the l
laacc operoperatorator,, which overlaps the which overlaps the transtranscripticription on start site, start site, bloc!bloc!s s transtranscriptcriptionion initiation b" the pol"merase.
initiation b" the pol"merase. When lactose is present, it binds to speci#c bindingWhen lactose is present, it binds to speci#c binding sit
sites in es in eaceach h subsubuniunit t of the of the tettetramrameriericc lalacc repressor, causing a conformationalrepressor, causing a conformational change in the protein that ma!es it dissociate from the
change in the protein that ma!es it dissociate from the laclac operator. As a result,operator. As a result, th
the e popol"l"memerarase se cacan n ininititiaiate te trtrananscscririptptioion n of of ththee llaacc operoperon. on. $owe$owever, ver, whenwhen glucose also is present, the rate of transcription initiation %i.e., the number of glucose also is present, the rate of transcription initiation %i.e., the number of times per minute di&erent pol"merase molecules initiate transcription' is ver" times per minute di&erent pol"merase molecules initiate transcription' is ver" lo
low, w, reresusultltining g in in s"s"ntnthehesisis s of of ononl" l" low low lelevevels ls ofof llaacc m()m()A A and and the the prproteoteinsins encoded in the
encoded in the laclac operon.operon. *n
*nce ce glglucucosose e is is dedeplpleteted ed frfrom om ththe e memedidia a anand d ththe e inintrtracacelellululalar r glglucucososee concentration falls,
concentration falls, E. coliE. coli cells respond b" s"nthesi+ing c"clic A, cA. As thecells respond b" s"nthesi+ing c"clic A, cA. As the concentration of cA increases, it binds to a site in each subunit of the dimeric concentration of cA increases, it binds to a site in each subunit of the dimeric CA protein, causing a conformational change that allows the protein to bind to CA protein, causing a conformational change that allows the protein to bind to the CA site in the
the CA site in the laclac transcription-control region. he bound CAcA comple/transcription-control region. he bound CAcA comple/ interacts with the pol"merase bound to the promoter, greatl" stimulating the rate interacts with the pol"merase bound to the promoter, greatl" stimulating the rate of transcrip
of transcription initiatition initiation. his on. his activactivation leads to ation leads to s"nths"nthesis of esis of high levels ofhigh levels of laclac m()A and subseuentl" of the en+"mes encoded b"
m()A and subseuentl" of the en+"mes encoded b" thethelaclac operon. operon.
0.
0. he conhe concencentratratiotion of n of frfree phosee phosphaphate a&ecte a&ects trants transcrscriptiption of someion of some E. coE. colili genes.genes. Describe the mechanism for this.
Describe the mechanism for this. Answer:
Answer:
When the phosphate concentration in the environment falls, it also falls in the When the phosphate concentration in the environment falls, it also falls in the periplasmic space, causing phosphate to dissociate from the ho( periplasmic periplasmic space, causing phosphate to dissociate from the ho( periplasmic domain, as depicted in igure 2-13. his causes a conformational change in the domain, as depicted in igure 2-13. his causes a conformational change in the ho( c"toplasmic domain that activates its protein !inase activit". he activated ho( c"toplasmic domain that activates its protein !inase activit". he activated ho( initiall" transfers a 4 -phosphate from A to a histidine side chain in the ho( initiall" transfers a 4 -phosphate from A to a histidine side chain in the ho( !inase domain itself. he same phosphate is then transferred to a speci#c ho( !inase domain itself. he same phosphate is then transferred to a speci#c aspartic ac
aspartic acid side id side chain in chain in ho5, convertinho5, converting ho5 g ho5 from afrom an n inactive to inactive to an activean active transcriptional activator. hosphor"lated, active ho5 then induces transcription transcriptional activator. hosphor"lated, active ho5 then induces transcription from several genes that help the cell cope
6n response to low phosphate concentrations in the environment and periplasmic space, a phosphate ion dissociates from the periplasmic domain of the inactive sensor protein ho(. his causes a conformational change that activates a protein !inase transmitter domain in the c"tosolic region of ho(. he activated transmitter domain transfers an A 4-phosphate to a conserved histidine in the transmitter domain. his phosphate is then transferred to an aspartic acid in the receiver domain of the response regulator ho5. everal ho5 proteins can be phosphor"lated b" one activated ho(. hosphor"lated ho5 proteins then activate transcription from genes encoding proteins that help the cell to respond to low phosphate, including phoA, phoS, phoE, and ugpB.
8. What is the evidence that transcriptional initiation is the primar" mechanism of gene control in comple/ organisms9
Answer:
Control of transcription initiation is the most important mechanism for determining whether most genes are e/pressed and how much of the encoded m()As and, conseuentl", proteins are produced. ()A processing and various post-transcriptional mechanism for controlling eu!ar"otic gene e/pression that resulting in gene control.
2. What t"pes of genes are transcribed b" ()A pol"merases 6, 66, and 6669 Design an e/periment to determine whether a speci#c gene is transcribed b" ()A pol"merase 66.
Answer:
- ()A pol"merase 6, located near nucleolus, transcribes genes encoding precursor r()A %pre-r()A', which is processed into 03, .3 and 13 r()As. - ()A pol"merase 66, transcibes all protein-coding genes; that is, it production
of m()As, also produce four of the #ve small nuclear ()As that ta!e part in ()A splicing.
- ()A pol"merase 666, transcribe genes encoding t()As, r()A, and an arra" of small stable ()As, including one involved in ()A splicing %<=' and the ()A component of the signal-recognition particle %(' involved in directing nascent proteins to the endoplasmic reticulum.
>/periment to determine whether a speci#c gene is transcribes b" ()A pol"merase 66:
Classes of ()A transcribe b" the three eu!ar"otic nuclear ()A pol"merases and ther functions
ol"merases ()A transcribe ()A function
()A pol"merase 6 re-r()A %03, .3 and 13 r()As'
(ibosome components, protein s"nthesis
()A pol"merase 66 m()A sn()As mi()As
>ncodes protein ()A splicing
ost trnscriptional gene control
()A pol"merase 666 t()As r()A sn()A <= 7 ()A
*ther stable short ()As
rotein s"nthesis
(ibosome copmponent, protein s"nthesis
()A splicing
ignal-recognition particle for insertion of pol"peptides into the >(
?arious function, un!nown for man"
. he CD of the largest subunit of ()A pol"merase 66 can be phosphor"lated and h"perphosphor"lated at various serine and t"rosine residues. What are the conditions that lead to phosphor"lation versus h"perphosphor"lation9
Answer:
CD %carbo/"l terminal domain'
- hospor"lation of CD occurs once the pol"merase initiates transcription and begins to move awa" from the promoter
- (emains phosphor"lated as the en+"me transcribe the template h"perphosphor"lated, preventing termination and permitting the pol"merase to contionue chain elongation
=. What do AA bo/es, initiators, and Cp@ islands have in common9 Which was the #rst of these to be identi#ed9 Wh"9
Answer:
- he three act as promoters in su!ar"oic D)A - Cp@
- a" contain transcription initiation region in its D)A
7. Describe the methods used to identif" the location of D)A control elements in regulator" regions of genes.
Answer:
D)A recombination
3. What is the di&erence between a promoter-pro/imal element and a distal enhancer9
Answer:
- romoter-pro/imal element:control regions lying within 100–200 base pairs upstream of the start site. In some cases, promoter-proximal elements are cell-type-specific; that is, they function only in specific differentiated cell types.
- Distal enhancer: control elements located thousands of base pairs away from the start site.
. Describe the methods used to identif" the location of D)A-binding proteins in the regulator" regions of genes.
Answer:
ootprinting and @el-hift Assa"s
1B. Describe the structural features of transcriptional activator and repressor proteins. Answer:
11. What happens to transcription of the EGR-1 gene in patients with Wilms tumor9 Wh"9
Answer:
10. <sing C(>5 and nuclear receptors as e/amples, compare and contrast the structural changes that ta!e place when these transcription factors bind to their co-activators.
Answer:
18. What structural change ta!es place on pol"merase 66 promoters during preinitiation comple/ formation9
Answer:
12. >/pression of recombinant proteins in "east is an important tool for biotechnolog" companies that produce new drugs for human use. 6n an attempt to get a new gene X e/pressed in "east, a researcher has integrated gene X into the "east genome near a telomere. Will this strateg" result in good e/pression of gene X 9 Wh" or wh" not9 Would the outcome of this e/periment di&er if the e/periment had been performed in a "east line containing mutations in the $8 or $2 histone tails9
Answer:
1. ou have isolated a new protein called 6CE. ou can predict from comparisons with other !nown proteins that 6CE contains a b$F$ domain and a in8-interacting domain. redict the function of 6CE and rationale for the importance of these domains in 6CE function.
Answer:
1=. Describe at least one gene "ou would e/pect to be able to clone using the following genes as bait in a "east two h"brid e/periment: alpha-globin; the catal"tic subunit of protein !inase A; and the catal"tic subunit of aspartate transcarbam"lase.
A)AFG> $> DAA
An electrophoretic mobilit" shift assa" %>A' was performed using a radiolabeled D)A fragment from the seuence upstream of gene X . his D)A probe was incubated with %H' or without %-' nuclear e/tract isolated from tissues A %bone'; 5 %lung'; C %brain'; and D %s!in'. he D)A: protein comple/es were then fractionated on nondenaturing pol"acr"lamide gels. he gels were e/posed to autoradiographic #lm; the results are presented in the #gure.
a. Which tissues contain a binding activit" that recogni+es the seuence upstream of gene X 9 6s the transcription factor the same in each tissue9
b. 6f the binding activit" was puri#ed, what test could be done to verif" that this factor is in fact a transcription factor9
c. What t"pe of assa" would be performed to determine the speci#c D)A seuence%s' to which the transcription factor binds9
d. 6f gene X is transcribed in lung and brain tissue but not in bone and s!in tissue, what t"pe of transcription factor is the binding activit"9 peculate as to the identit" of other factors that might be comple/ed at the gene X promoter in bone and s!in tissue.
