Re iew
One s ep back be o e mo ing o wa d: Regula ion o ansc ip ion elonga ion
by a es and back acking
Fe nando Gómez-He e os, Lola de Miguel-Jiménez, Gonzalo Millán-Zamb ano, Xenia Peña e,
Lidia Delgado-Ramos, Ma i C uz Muñoz-Cen eno, Sebas ián Chá ez
⇑
Depa amen o de Gené ica, Uni e sidad de Se illa, Se ille, Spain
a icle in o
A icle his o y:
Recei ed 4 July 2012
Re ised 9 July 2012
Accep ed 10 July 2012
A ailable online 17 July 2012
Edi ed by Miguel De la Rosa, Felix Wieland
and Wilhelm Jus
Keywo ds:
RNA polyme ase II
T ansc ip ion elonga ion
Back acking
TFIIS
RNA clea age
Ribosomal p o ein genes
abs ac
RNA polyme ase II back acking is a well-known phenomenon, bu i s in ol emen in gene egula-
ion is ye o be add essed. S uc u al s udies in o he back acked complex, new eac i a ion mech-
anisms and genome-wide app oaches a e shedding some ligh on his in e es ing aspec o gene
ansc ip ion. In his e iew, we b iefly summa ise hese new findings, commen abou some esul s
ecen ly ob ained in ou labo a o y, and p opose a new model o he influence o he ch oma in
con ex on RNA polyme ase II back acking.
1. In oduc ion
RNA polyme ase II plays a key ole in he exp ession o euka y-
o ic genomes. All p o ein-encoding genes a e ansc ibed by his
polyme ase, whose s uc u al and unc ional p ope ies enable
he g ea di e si y o egula o y p og ams ha a e execu ed
h oughou he genome. The bes -known egula ion mechanisms
ope a e du ing p e-ini ia ion complex assembly and ini ia ion,
bu a significan numbe o genes egula e hei ansc ip ion in
pos -ini ia ion s eps ( e iewed in [1]). Among o he s, he mecha-
nisms sus aining elonga ion should p o ide he oppo uni y o eg-
ula e ansc ip ion.
T ansc ip ion elonga ion is a om a uni o m p ocess. RNA
polyme ase II p ofiles, measu ed by ch oma in immunop ecipi a-
ion (ChIP), ansc ip ional un-on o nascen RNA sequencing ha e
es ablished clea di e ences be ween genes wi hin he same gen-
ome [2–5]. Single-cell expe imen s ha e also shown ha RNA
polyme ase II p og ession along a ansc ibed gene is a discon in-
uous p ocess which combines sho ad ances wi h pauses o a a -
iable ime ex en [6]. All hese esul s demons a e ha pausing is
a equen phenomenon ha is likely consubs an ial o RNA poly-
me ase II-dependen ansc ip ion.
In i o expe imen s ha e demons a ed ha RNA polyme ase II
pausing is a highly uns able s a e which esul s in ei he o wa d
ansc ip ion o s able a es [7]. RNA polyme ase II a es in ol es
back acking, a e e se mo emen ha b ings abou loss o con ac
be ween he 3
0
end o nascen RNA and he RNA polyme ase II ac-
i e si e. As a esul o his mislocalisa ion, back acked RNA poly-
me ases canno esume ansc ip ion immedia ely. The e na y
complex o med by DNA, RNA and back acked RNA polyme ase
II is ex emely s able. Recen s uc u al da a published by C ame ’s
lab explain his s abili y by he specific binding o eigh nucleo ides
o back acked RNA o a highly conse ed si e in he RNA polyme -
ase II po e and unnel, which aps a p o ein loop loca ed in he ac-
i e si e [8].
In i o and in i o s udies ha e ound a la ge numbe o causes
which b ing abou RNA polyme ase II a es . The sca ci y o nucle-
o ides a ou s RNA polyme ase II a es in i o. D ugs ha p o-
oke deple ion o NTP pools, like 6-azau acil and mycophenolic
acid, lowe he elonga ion a e and he p ocessi i y o RNA poly-
me ase II in i o [9], which inc eases equency o a es [10].
Yeas mu an s lacking RNA polyme ase II eac i a ion ac o s, like
TFIIS o Cc 4-No (see la e ), exhibi hype sensi i i y o hese NTP-
deple ing d ugs.
h p://dx.doi.o g/10.1016/j. ebsle .2012.07.030
⇑
Co esponding au ho . Add ess: A da Reina Me cedes 6, E41012 Se ille, Spain.
Fax: +34 954557104.
E-mail add ess: [email p o ec ed] (S. Chá ez).
FEBS Le e s 586 (2012) 2820–2825
jou nal homepage: www.FEBSLe e s.o g
Ó2012 Fede a ion o Eu opean Biochemical Socie ies. Published by Else ie B.V.
Open access unde CC BY-NC-ND license.
0014-5793 Ó2012 Fede a ion o Eu opean Biochemical Socie ies. Published by Else ie B.V.
Open access unde CC BY-NC-ND license.
A e y in ui i e cause o RNA polyme ase II a es is he p es-
ence o obs acles in DNA. Among hem, nucleosomes a e he mos
ubiqui ous s uc u es o hinde euka yo ic ansc ip ion. In i o
s udies ha e clea ly shown ha nucleosomes p omo e back ack-
ing and ha ansc ip ion o nucleosomal empla es is s imula ed
by TFIIS, his being he RNA clea age ac o ha eac i a es back-
acked RNA polyme ase II [11](see la e ).
T ansc ip ion elonga ion gene a es o sional s ess on DNA,
which needs o be emo ed by oposiome ases [12]. This accumu-
la ion o posi i e supe coiling is pa icula ly de imen al o an-
sc ip ion elonga ion h ough yeas genes longe han 3 kpb [13].
Topological cons ains is, he e o e, ano he po en ial sou ce o
RNA polyme ase II s alling. In e es ingly, he impac o DNA o -
sional s ess on ansc ip ion is condi ioned by he ch oma in o ga-
nisa ion o he ansc ip ion uni , in a double way. On one hand,
ch oma in compac ion acili a es RNA polyme ase II elonga ion
[14]; on he o he hand, nucleosome ee egions acili a e opoiso-
me ase II ec ui men o ansc ibed genes [15].
DNA lesions a e cause o RNA polyme ase a es as well. Bulky
DNA adduc s wi hin ansc ibed egions lead o RNA polyme ase
II a es . In mos cases his a es is i e e sible and needs o be
sol ed by he deg ada ion o he s alled polyme ase ( e iewed in
[16]). Undamaged DNA sequences can also a ou RNA polyme ase
II a es in i o, e en when he empla e akes a non-nucleosomal
configu a ion [17]. Py imidine- ich acks in he non- empla e
s and, like he TTTTTTTCTCCATTTT sequence p esen in he in in-
sic ‘‘ e mina o ’’ egion wi hin he c-myc fi s exon–in on bound-
a y, induce RNA polyme ase II a es [18]. This sequence-
dependen a es was fi s explained by he esul s o he weak
DNA–RNA in e ac ions caused by he high p opo ion o U = A
pai s. La e , howe e , esul s indica e ha he p esence o cy o-
sines in he polypy imidine ack is no de imen al o a es
[19]. This is in good ag eemen wi h he s ong p e e ence o
py imidines ound in he specific in e ac ion be ween back acked
RNA and he ‘‘back acked si e’’, in he RNA polyme ase II po e and
unnel [8]. This sequence specifici y es ablishes he basis o an
a es ing code ac oss he genome, which migh explain he biased
dis ibu ion o he polypy imidine/polypu ine acks be ween em-
pla e and non- empla e s ands [20].
2. Molecula mechanisms o e coming RNA polyme ase II a es
RNA polyme ase a es canno be pe manen , since his would in-
ol e he comple e ep ession o gene exp ession and would se i-
ously hinde DNA eplica ion, leading o genome ins abili y and
e en ually o cell dea h ( e iewed in [16]). E en in hose cases whe e
RNA polyme ase a es plays a ole in gene egula ion, he mecha-
nisms capable o esuming ansc ip ion o emo ing a es ed poly-
me ase a e equi ed o ansc ip ion cycle iabili y. Th ee di e en
mechanisms ope a ing in a es ed RNA polyme ase II ha e been de-
sc ibed: RNA clea age, allowing he RNA 3
0
end o eloca e a he ac-
i e si e; e e sion o he back acked s a e by he o wa d
mo emen o he polyme ase; and e ic ion o a es ed RNA poly-
me ase II by ubiqui yla ion-media ed deg ada ion (Fig. 1).
Endonucleoly ic ac i i y allows 3
0
RNA clea age, which is an
inhe en p ope y o RNA polyme ase II ha becomes highly s im-
ula ed by clea age ac o TFIIS [21]. The s uc u al basis o TFIIS-
assis ed RNA clea age has been ecen ly explained [8]. TFIIS do-
main III ex ends in o he RNA polyme ase II po e, eaching he ac-
i e si e and emodels he RNA polyme ase II mo i s ha bind
back acked RNA. This enables he displacemen o RNA om he
back acked si e. In addi ion, TFIIS complemen s he ac i e si e
wi h se e al esidues ha may ca alyze p o on ans e s du ing
RNA clea age [8].
E en in he absence o TFIIS, a es ed RNA polyme ase II can be
eac i a ed by RNA clea age. This has been elegan ly shown by
S esj up’s lab using a dominan nega i e TFIIS mu an ha can
bind a es ed RNA polyme ase II wi hou s imula ing RNA clea -
age. The esul ing e na y complex s abilises he back acked con-
figu a ion by inhibi ing spon aneous RNA clea age [22]. The le hal
pheno ype o his mu an in yeas , unlike he iabili y o he TFIIS
dele ion mu an , indica es ha RNA polyme ase II a es is a e y
common phenomenon which yeas cells unde s anda d g owing
condi ions can sol e by ei he spon aneous o TFIIS-s imula ed
RNA clea age, o e en by al e na i e mechanisms.
One o hese al e na i e mechanisms is media ed by Cc 4-No .
This is an e olu iona ily conse ed complex composed o nine sub-
uni s. Based on independen expe imen al app oaches, Cc 4-No
has been connec ed o di e en aspec s o gene ansc ip ion,
including ini ia ion [23,24] and elonga ion [2,25,26]. Cc 4 is he
majo cy oplasmic mRNA deadenylase in yeas and he complex
localises o p ocessing bodies whe e mRNA deg ada ion akes
place [27,28]. In his mul i unc ionali y con ex , Reese’s lab has
demons a ed ha Cc 4-No s imula es ansc ip ion elonga ion
by p omo ing he esump ion o elonga ion by a es ed polyme -
ases [29]. The in e ac ion o Cc 4-No wi h he eme ging ansc ip
is equi ed o his way o eac i a ing a es ed RNA polyme ase II,
which does no in ol e RNA clea age (Fig. 1).
The desc ibed eac i a ion mechanisms a e likely o o e come
mos RNA polyme ase II a es e en s, bu cells ha e an addi ional
way o con ending wi h i e e sibly blocked ansc ip ional com-
plexes, namely deg ada ion [30,31]. This mechanism in ol es
ubiqui yla ion o he bigges RNA polyme ase II subuni , Rpb1, a
di e en si es in a p ocess ha is specifically media ed by he ubiq-
ui yla ion ac o De 1 and he CTD domain o Rpb1 [32]. Mu a ions
a ec ing Rpb1 ubiqui yla ion and RNA clea age a e syn he ic
le hal, indica ing ha RNA clea age o deg ada ion a e he only
possible al e na i es, a leas o a subse o a es ed RNA polyme -
ase II molecules [32]. In addi ion o he p o easome-dependen
deg ada ion o RNA polyme ase II, Rpb1 ubiqui yla ion migh also
p omo e o he al e na i e esump ion p ocesses p io o deg ada-
ion (see he discussion o his issue in [33]). In ligh o his, i is
meaning ul ha De 1 is an in e ac ing ac o o Rad26, a DNA heli-
case in ol ed in ansc ip ion-couple epai , which inhibi s RNA
polyme ase II deg ada ion [34]. A scena io, in which Rpb1 ubiqui-
yla ion p omo es he disassembly o a es ed RNA polyme ase II
in a helicase-assis ed way, is an a ac i e possibili y ha emains
o be expe imen ally es ed (Fig. 1).
Fig. 1. Mechanisms esol ing RNA polyme ase II back acking. RNA polyme ase II
back acking in ol es he eloca ion o he 3
0
end o nascen RNA ou side he ac i e
si e. This can be sol ed spon aneously by he in insic RNA clea age ac i i y o he
enzyme (a) bu i ge s s ongly s imula ed by TFIIS (b). Al e na i ely Cc 4, a subuni
o he Cc 4-No complex, can s imula e o wa d- acking, hus allowing RNA
polyme ase II o esume elonga ion in an RNA clea age-independen manne (c).
A es ed RNA polyme ase II can be emo ed om DNA by ubiqui yla ion and i s
subsequen deg ada ion by he p o easome (d). The e na y complex o med by
DNA, RNA and he back acked RNA polyme ases migh also be disassembled,
in ol ing ansc ip ion e mina ion (e). Howe e no clea expe imen al e idence is
a ailable o his mechanism.
F. Gómez-He e os e al. / FEBS Le e s 586 (2012) 2820–2825 2821
3. Gene egula ion by RNA polyme ase II a es
T ansc ip ional egula ion in pos -ini ia ion s ages is a common
phenomenon ac oss me azoan genomes. A leas 40% o genes
show p omo e -p oximal accumula ion o RNA polyme ase II in
mammalian, D osophila and Caeno habdi is cells [3,35–39].
An example o egula ion a he le el o elonga ion is ound in
he so-called bi alen genes in emb yonic s em cells. They display
cha ac e is ic ch oma in ma ke s o silenced (his one H3K27 i-
me hyla ion) and ac i e ansc ip ion (his one H3K4 i-me hyla-
ion)[40]. A subg oup o bi alen genes, bound by Polycomb
ep essi e complex 2, displays s alled RNA polyme ase II. Since s a-
ble pausing leads o RNA polyme ase II a es , i is likely ha he
RNA polyme ase II molecules p esen in silenced bi alen genes
a e a es ed; howe e , he e is no di ec expe imen al e idence
o his hypo hesis. Such e idence ac ually exis s o ano he g oup
o mammalian genes which exhibi p omo e -p oximal accumula-
ion o RNA polyme ase II. G ow h-p omo ing, p o-oncogenic
genes like FOS and MYC eside p e e en ially in compac ch oma in
and a e ine ficien ly ansc ibed unde basal condi ions. The an-
sc ip ion o hese genes is ep essed du ing ea ly elonga ion by a
mechanism in e e ing wi h TFIIS ec ui men ha in ol es H2B
ubiqui yla ion [41].
The in ol emen o TFIIS is also demons a ed in he ac i a ion,
in esponse o hea shock, o he s alled RNA polyme ases occupy-
ing he p omo e p oximal egion o D osophila Hsp70 [42]. Using
RNA in e e ence, in i o TFIIS deple ion p o okes a delay in Hsp70
induc ion which, in his case, is only possible a e a new ound o
ansc ip ion ini ia ion [42]. Acco dingly, we can conclude ha
mos , i no all, s ably s alled RNA polyme ases would be back-
acked and would equi e TFIIS o any al e na i e eac i a ion
mechanism o eco e he elonga ion capabili y upon ac i a ion.
One di ec way o de ec ing back acked RNA polyme ases is o
compa e he genomic pa e ns o un-on signals, which eflec
elonga ion-p oficien RNA polyme ases [43], wi h he genomic dis-
ibu ion o he o al RNA polyme ase II measu ed by ChIP. This
s a egy de ec ed clea di e ences be ween he wo pa e ns
ac oss he yeas genome [44]. The e is a gene al high co ela ion
be ween un-on and ChIP signals, bu some gene on ology ca ego-
ies exhibi lowe un-on alues han expec ed acco ding o hei
ChIP signals [44]. This di e ence p o es pa icula ly s iking o
hose genes encoding ibosomal p o eins (RP), indica ing he accu-
mula ion o back acked RNA polyme ase II in hese genes. One
pa icula beha iou o RNA polyme ase II du ing ansc ip ion
elonga ion o RP genes is also eflec ed by he 5
0
-biased dis ibu-
ion o he un-on signals in his specific g oup o genes [2]. This
bias was also obse ed in he dis ibu ion o nascen RNA o highly
exp essed genes ac oss he yeas genome [5]; he high p opo ion
o highly ansc ibed genes belonging o he RP g oup migh ex-
plain his esul .
The un-on signals o RP genes lowe ed when compa ing yeas
cells exponen ially g owing in glucose-con aining medium o cells
exponen ially g owing in galac ose-con aining medium. Howe e ,
his dec ease was significan ly mo e ma ked in he o al RNA poly-
me ase II le els de ec ed by ChIP han in he un-on signals;
acco dingly, he un-on/ChIP a io o he RP genes inc eased om
glucose o galac ose (Fig. 2). The nuclea genes encoding mi ochon-
d ial componen s exhibi ed he opposi e change (Fig. 2)[44]. The
simples in e p e a ion o hese esul s in ol es a specific educ-
ion in he numbe o back acked RNA polyme ase II complexes
in esponse o he ca bon sou ce (Fig. 2). In ac , he un-on/ChIP
a io o RP genes depends on he in eg i y o he Ras-PKA pa hway
and on he silencing domain o Rap1, an essen ial ansc ip ion ac-
o o RP genes ansc ip ion [44]. In sho , hese obse a ions sug-
ges ha RNA polyme ase II back acking is an impo an elemen
in he ansc ip ional egula ion o RP genes and o o he gene am-
ilies ac oss he yeas genome.
A egula o y mechanism based on RNA polyme ase II back ack-
ing, o cons i u i ely exp essed genes like RP, mus in ol e an e fi-
cien eac i a ion mechanism. A se ies o expe imen s ca ied ou
in ou labo a o y demons a e ha TFIIS is pa icula ly in ol ed
in he eac i a ion o back acked RNA polyme ase II on RP genes,
a leas unde he s ong ansc ip ional s ess caused by 6AU [45].
Sho ly a e adding his d ug o yeas cells, RNA polyme ase II
complexes ansc ibing he RP genes become en iched in TFIIS, in
compa ison o he o he highly ansc ibed genes es ed, which
do no ela e o ibosomes (Fig. 3A). This di e ence be ween RP
and non-RP genes is pa icula ly s iking in he 5
0
end o he an-
sc ibed egion, whe e RNA polyme ase II accumula es upon NTP
deple ion [9]. RP genes main ain high TFIIS/Rpb3 a ios in 5
0
upon
6AU ea men , whe eas in non-RP genes RNA polyme ase II shi s
owa ds he 5
0
egion while TFIIS does no , esul ing in low TFIIS/
Rpb3 a ios (Fig. 3A). The low TFIIS demand by paused RNA poly-
me ase II in non-RP genes an icipa es a lowe le el o back acking
han in RP genes, which is confi med by he ansc ip ional un-on
assays (Fig. 3B). In ac , he compa ison o wild- ype and ds 1
D
(TFIIS-dele ed) s ains shows ha he un-on/RNA polyme ase II
ChIP a ios o RP genes in he p esence o 6AU a e s ic ly depen-
den on TFIIS, whe eas he e ec o 6AU on non-RP genes’ un-
on/RNA polyme ase ChIP a ios is much milde and does no de-
pend on TFIIS o a simila ex end (Fig. 3B).
We ha e also showed ha his high dependence o yeas RP
gene ansc ip ion on TFIIS can be supp essed by dele ing genes
encoding ansc ip ional egula o s o RP gene exp ession, like
S p1 [45]. In he absence o S p1, RP genes do no depend on TFIIS
o main ain ac i e elonga ing RNA polyme ase molecules on hei
gene bodies (Fig. 3B). Mo eo e , he absence o S p1 supp esses
RNA polyme ase II accumula ion on he 5
0
end in all es ed genes,
sugges ing ha his p o ein plays a gene al ole in p omo ing RNA
polyme ase II pausing [45].
By way o conclusion, he specific egula ion imposed by ac o s
like S p1 o yeas RP genes du ing elonga ion, in ol ing RNA poly-
me ase II back acking, makes his g oup o genes mo e dependen
on eac i a ion ac o s, like TFIIS, o esis ansc ip ional s ess.
4. Back acking in he ch oma in con ex
The mechanism explaining his di e en ial endency o RNA
polyme ase II o back ack is unknown. I may be due o a di e en-
ial composi ion o RNA polyme ase II elonga ion machine y,
he eby imposing di e en modes o ansc ip ion o specific genes
o egulons. A biased composi ion o RNA polyme ase II i sel is an
appealing hypo hesis. The Rpb4/7 submodule has been shown o
d op easily om RNA polyme ase II in i o and o play indepen-
den oles in i o [46]. pb4
D
mu an s display syn he ic le hali y
wi h ds 1
D
,lacking TFIIS, and a e ex emely sensi i e o NTP-
deple ing d ugs [26,47]. I would be, he e o e, concei able ha
in some genes RNA pol II was p one o back acking as a esul o
a p ema u e d op o Rpb4/7 du ing elonga ion. Expe imen al da a,
howe e , do no suppo his hypo hesis so a . The Rpb3/Rpb7 a-
io is la gely in a iable ac oss he yeas genome [48]. Mo eo e ,
he composi ion o he whole RNA polyme ase elonga ion machin-
e y is la gely cons an o all yeas genes [49], sugges ing ha he
di e en ial back acking beha iou is likely due o ansc ibed
genes a he han o ansc ip ional machine y.
In con as , ch oma in is ma kedly polymo phic ac oss he gen-
ome and nucleosome posi ioning is no uni o m. Yeas RP genes,
o ins ance, show sho e nucleosome epea han non-RP genes
[50]. His one a ian s and ch oma in co alen modifica ions also
display gene- o-gene a ia ion ac oss he genome [51]. Acco d-
2822 F. Gómez-He e os e al. / FEBS Le e s 586 (2012) 2820–2825
ingly, he di e en ial unc ional in e ac ion be ween RNA poly-
me ase II and he gene-specific configu a ion o ch oma in migh
explain i s di e en ial endency o back ack. I his hypo hesis
we e ue, elonga ion h ough some ch oma in configu a ions
would ake place wi hou back acking, whe eas o he al e na i e
ch oma in configu a ions would be highly p one o back acking.
In he la e , TFIIS would be highly demanded. In i o expe imen s
pe o med in Kashle ’s labo a o y ha e shown ha TFIIS is indeed
equi ed o RNA polyme ase II o o e come he s ong s alling im-
posed by a nucleosome, whe eas he same DNA empla e, when
naked, does no p omo e RNA polyme ase II pausing o equi es
TFIIS [52]. The combina ion o TFIIS and TFIIF, ano he gene al an-
sc ip ion ac o ha s imula es elonga ion, syne gis ically s imu-
la e nucleosome ansc ip ion by RNA polyme ase II [53].
In hese la e expe imen s, nucleosome a e sal by RNA poly-
me ase II ook place wi hou nucleosome displacemen . S udi sky’s
labo a o y has shown ha nucleosomes can indeed su i e an-
sc ip ion in i o, by allowing he o ma ion o an in anucleosomal
DNA loop (Ø loop) ha con ains he ansc ibing enzyme [54]. This
Ø loop likely equi es specific DNA-his one in e ac ions, since his-
one Sin mu a ions, dis up ing his kind o in e ac ions, comp o-
mise nucleosome su i al du ing elonga ion [55].
Recen esul s ob ained by a omic o ce mic oscopy also indi-
ca e ha RNA polyme ase II can ansc ibe a nucleosome wi hou
p omo ing i s comple e disassembly, bu jus emo ing a single
H2A-H2B dime [56]. A simila conclusion was ob ained by analys-
ing he a e o nucleosomal his ones du ing in i o ansc ip ion: a
emodelled nucleosome, deple ed o a single H2A-H2B dime by
his one chape one Nap1 ac ion, can be ansc ibed wi hou com-
ple e his one e ic ion [57]. In ag eemen wi h hese esul s, his-
one hexasomes (a H3-H4 e ame associa ed o a single H2A-
H2B dime in he con ex o a emodelled nucleosome [58]) ha e
long since been ela ed o ansc ibed ch oma in [59].
In his eme ging model, RNA polyme ase II would be able o
elonga e ansc ip ion along a nucleosomal DNA empla e by ak-
ing ad an age o he Ø loop ha he missing H2A-H2B dime
would p o oke wi hin he nucleosome. I is di ficul o imagine
such a way o ansc ibing nucleosomal DNA as a con inuous un,
Fig. 2. The p opo ion o ac i e, un-on-compe en RNA polyme ase II is egulon-specific and con olled by he yeas cell in esponse o physiological s imuli. Yeas ibosomal
p o ein genes show a significan p opo ion o elonga ing RNA polyme ase II molecules ha do no p oduce a ansc ip ional un-on signal ( ed) when exponen ially g owing
in glucose-con aining medium. The same genes, when cells a e exponen ially g owing in galac ose-con aining medium, show lowe le els o elonga ing RNA polyme ase II
bu exhibi a much highe p opo ion o un-on compe en enzymes (g een). The yeas genes encoding mi ochond ial elemen s exhibi he opposi e egula ion: low
p opo ion o ac i e polyme ase in galac ose medium and high p opo ion, al hough limi ed le els, in glucose medium. Mos o yeas genes do no exhibi a significan
p opo ion o un-on-incompe en cells unde ei he o he wo g ow h condi ions. Adap ed om he expe imen al da a desc ibed in [44].
Fig. 3. TFIIS is equi ed pa icula ly o main ain RNA polyme ase II ac i i y on ibosomal p o ein genes unde ansc ip ional s ess. A. A 15 min 6AU ea men causes a 5
0
-
shi in he dis ibu ion o RNA polyme ase II along he ansc ibed egion in wild- ype yeas cells. A he same ime, RP genes dec ease a he absolu e le els o RNA
polyme ase II, which eflec s hei down- egula ion in esponse o he g ow h impai men caused by he d ug. TFIIS dis ibu ion pa allels ha o RNA polyme ase II in RP
genes. In non-RP genes, TFIIS does no unde go he 5
0
-shi o RNA polyme ase II upon 6AU ea men . B. Compa ison o he wild- ype and ds 1
D
un-on pa e ns allow us o
conclude ha RP genes equi e TFIIS o main ain he ac i i y o hei RNA polyme ase molecules upon 6AU addi ion. Non-RP genes show a much milde dependence on TFIIS
unde he same condi ions. This di e en ial beha iou o RP and non-RP genes depends on egula o y ac o S p1. In bo h s p1
D
and s p1
D
ds 1
D
cells, RNA polyme ase
ac i i y and in agenic dis ibu ion a e no influenced by 6AU. Adap ed om he expe imen al da a desc ibed in [45].
F. Gómez-He e os e al. / FEBS Le e s 586 (2012) 2820–2825 2823
bu ins ead as a discon inuous phenomenon in ol ing al e na i e
ansc ip ion elonga ion and his ones-DNA econfigu a ion s eps
a ound he nucleosome. In his con ex , equen RNA polyme ase
II back acking would be expec ed (Fig. 4). A compa ison made o
he massi e sequencing o nascen RNA wi h high esolu ion nucle-
osome posi ioning showed a paused dis ibu ion o elonga ing RNA
polyme ase II wi hin nucleosomes [60], which is consis en wi h
his hypo hesis. In con as , a ansc ip ional elonga ion mode
domina ed by his one e ic ion and comple e nucleosome disas-
sembly migh p o e mo e simila o in i o naked DNA ansc ip-
ion and, he e o e, migh be less p one o back acking, and less
TFIIS-dependen (Fig. 4).
Gene-specifici y in back acking migh a ise om hese al e na-
i e modes o ansc ip ion elonga ion. In some cases, as in he RP
egulon, he hexasome-media ed, TFIIS-dependen elonga ion o
nucleosomal DNA would be dominan . In o he cases, such as
SAGA-dependen inducible genes, comple e his one e ic ion would
be dominan and ansc ip ion elonga ion would depend much less
on TFIIS and be less p one o back acking. The obse a ion o posi-
ioned nucleosomal p ofiles being mo e esis an o ansc ip ion in
RP genes han in SAGA-dependen genes suppo s his model [51].
The exis ence o wo al e na i e modes o ansc ip ion elonga-
ion, depending on he ch oma in con ex , can help explain he ia-
bili y o yeas mu an s lacking TFIIS. In hem, he his one e ic ion
mode would escue RNA polyme ase II om gene al a es . In his
con ex , hose egula o y mechanisms p omo ing he hexasome-
media ed mode would enhance he dependence o RNA polyme -
ase II on TFIIS and o he eac i a ing ac o s. Fac o s like yeas
S p1 fi in his egula o y ole (Fig. 4).
5. Rema ks
RNA polyme ase II pausing du ing elonga ion is a common phe-
nomenon. The connec ion be ween pausing and back acking dem-
ons a ed in i o and he in ol emen o TFIIS in he egula ion o
some genes du ing elonga ion sugges ha RNA polyme ase II
back acking is likely an impo an elemen in euka yo ic gene
con ol. The conse ed mo i es in ol ed in s abilising back acked
RNA inside RNA polyme ase II ma ch his gene al back acking ole
in gene egula ion.
The compa isons made be ween he un-on and RNA polyme -
ase II ChIP p ofiles in yeas a e compa ible wi h a significan p o-
po ion o back acked RNA polyme ases in ce ain gene amilies;
e.g., RP genes. The ansc ip ional egula ion o RP genes, in e-
sponse o changes in he ca bon sou ce o he medium, in ol es
changes in he p opo ion o back acked RNA polyme ases. This
egula o y mechanism is media ed by ansc ip ion ac o Rap1.
Back acking equi es ei he eac i a ion o RNA polyme ase II
emo al mechanisms. Reac i a ion can be achie ed by RNA clea -
age, a p ocess ha is s ongly s imula ed by TFIIS. Reac i a ion can
also be media ed by he Cc 4-No complex in a p ocess ha does
no in ol e RNA clea age. Fo RP genes, TFIIS is equi ed o esis
he ansc ip ional s ess imposed by NTP-deple ing d ugs like
6AU. This equi emen depends on he egula o y p og amme im-
posed by specific ansc ip ion ac o s, like S p1.
Wha makes some genes pa icula ly p one o RNA polyme ase II
back acking emains unknown, bu he eme ging models p oposed
o explain ch oma in ansc ip ion may help shed some ligh on his
phenomenon. The p ecise cha ac e isa ion o he mechanisms egu-
la ing RNA polyme ase II back acking is, he e o e, one o he o h-
coming challenges in he ansc ip ion esea ch field.
Acknowledgmen s
This wo k has been suppo ed by he Minis y o Economy and
Compe i i eness (G an s BFU2007-67575-C03-02, BFU-2010-
21975-C03-03 o S. Ch., FPI ellowships o L. de M. and L.D.-R.,
and FPU ellowship o G.M.-Z.), he Andalusian Go e nmen
(G an s P07-CVI-02623 and P08-CVI-03508), and by he Eu opean
Union (Regional De elopmen Eu opean Fund). We hank Helen
Wa bu on o English co ec ions.
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