Chapter 7 1. Descri Describe be the molec molecula ularr events events that that occur occur at the lac operon when E. coli cells are shifted from a glucose-containing medium to a lactose-containing medium. Answer: When no lactose is present, binding of the lac repressor to a seuence called the lac oper operator ator, which overlaps the trans transcripti cription on start site, bloc! bloc!s s trans transcript cription ion initiation b" the pol"merase. When lactose is present, it binds to speci#c binding sites sit es in eac each h sub subuni unitt of the tet tetram rameri eric c la lac c repressor, causing a conformational change in the protein that ma!es it dissociate from the lac operator. As a result, the th e po pol" l"me mera rase se ca can n in init itia iate te tr tran ansc scri ript ptio ion n of th the e lac oper operon. on. $owe $owever, ver, when 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" low, lo w, re resu sult ltin ing g in s" s"nt nthe hesi sis s of on onl" l" lo low w le leve vels ls of lac m() m()A A and the pr prote oteins ins encoded in the lac operon. *nce *n ce gl gluc ucos ose e is de depl plet eted ed fr from om th the e me medi dia a an and d th the e in intr trac acel ellu lula larr gl gluc ucos ose e concentration falls, E. coli cells 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 CA protein, causing a conformational change that allows the protein to bind to the CA site in the lac transcription-control region. he bound CAcA comple/ interacts with the pol"merase bound to the promoter, greatl" stimulating the rate of transcription transcription initiation. initiation. his activ activation ation leads to s"nth s"nthesis esis of high levels of lac m()A and subseuentl" of the en+"mes encoded b" the lac lac operon. operon.
0. he concen concentra tratio tion n of fr free ee phospha phosphate te a&ects a&ects transcr transcript iption ion of some E. coli coli genes. Describe the mechanism for this. Answer: When the phosphate concentration in the environment falls, it also falls in the periplasmic space, causing phosphate to dissociate from the ho( periplasmic 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( 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 aspartic acid acid side chain in ho5, converting converting ho5 from an an inactive to an active transcriptional activator. hosphor"lated, active ho5 then induces transcription from several genes that help the cell cope with low phosphate conditions.
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 (ibosome components, 13 r()As' protein s"nthesis ()A pol"merase 66
m()A sn()As mi()As
()A pol"merase 666
t()As r()A sn()A <= 7 ()A
*ther stable short ()As
>ncodes protein ()A splicing ost trnscriptional gene control 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. 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 preinitiation comple/ formation9
on pol"merase 66 promoters
during
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 in8interacting 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. Answer:
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. Answer:
