biomol transe

7/24/2019 biomol TRANSE

1/40

INDONESIANYA DI BAWAHTAPI MASIH ACAK ACAKANMAAF

Initiation of transcriptionThe basic echanis of itochon!ria" transcription

has been so"#e! in representati#e species of se#era"ph$"o%enetic %ro&ps 're#ie(e! in )*+,*-./0 H&antDNA transcription initiation sites an!prooter re%ions ha#e been !eterine! &sin% a #ariet$of techni1&es2 inc"&!in% 3P4en! appin% of priar$itochon!ria" transcripts b$ S5 n&c"ease protectione6perients )*7,85. an! !e"etion )89,88.2 site!irecte!&ta%enesis )83. an! "in:er s&bstit&tionana"$ses )8+. of c"one! tDNA fra%ents in r&no; transcription assa$s0 A"" a#ai"ab"e !ata areconsistent (ith the conc"&sion that there are t(oa


2/40

)*7,8528*.0A"tho&%h there is a fair"$ !etai"e! pict&re of thecis4actin% e"eents in#o"#e! in tDNA transcription2:no("e!%e of the trans4actin% n&c"ear4enco!e!factors is sti"" incop"ete0 Bioph$sica" fractionationof h&an itochon!ria" transcription e6tracts ha#e

re#ea"e! the re1&ireent of at "east t(o trans4actin%proteins a re"ati#e"$ non4se"ecti#e core NA po"$erasean! a !issociab"e transcription factor (hichconfers prooter se"ecti#it$ on the po"$erase)823>.0 The core en$e is e6pecte! to interact(ith the HSP an! =SP as &tations in these !oainsob"iterate e#en the "o(est "e#e" of transcription)8+.0 Mitochon!ria" NA po"$erases ha#e not beenp&rie! to hoo%eneit$0 Ne#erthe"ess2 h&an cDNAspecif$in% itochon!ria" NA po"$erase (as recent"$i!entie! b$ screenin% of an e6presse! se1&enceta%s 'EST/ !atabase (ith the $east se1&ence

)35.0 Interestin%"$2 the C4terina" ha"f of the pre!icte!po"$pepti!e shares si%nicant aino aci! se1&encei!entit$ (ith the sin%"e s&b&nit NA po"$erasesof T*2 T an! SP+ bacteriopha%es0

The h&an !issociab"e transcription factor actin%in concert (ith the core itochon!ria" NA po"$erasehas been p&rie! )3923*.2 its cDNA has beenc"one! an! se1&ence! )38. an! its %ene has beencharacterise! )33,3.0 The factor2 no( tere!tTFA )3-.2 is an ab&n!ant 934:Da itochon!ria"protein an! is "ar%e"$ coprise! of t(o hi%h obi"it$%ro&p 'HM?/ !oains separate! b$ a 94aino aci!

resi!&e "in:er an! fo""o(e! b$ a 934aino aci! resi!&ebasic C4terina" tai"0 HM? !oains are consi!ere!to be in#o"#e! in DNA bin!in% an! arefo&n! in a rather !i#erse fai"$ of proteins (hoseebers ha#e been ip"icate! in processes s&ch astranscription enhanceent an! chroatin pac:a%in%)37.0 M&tation ana"$sis of the h&an tTFA has!eonstrate! that its C4terina" tai" is iportantfor specic DNA reco%nition an! is essentia" forsponsorin% of hi%h "e#e"s of specic initiation of transcription)+>.0In #itro DNase I protection st&!ies in cobination

(ith r&n4o; transcription assa$s ha#e !eonstrate!that bin!in% of tTFA to re%ions ie!iate"$ &pstreaof the HSP or =SP '5>,8> bp &pstrea ofeach start site/ is re1&ire! for specic initiation oftranscription )8+2823923*.0 Bin!in% of tTFA atthese re%ions (as recent"$ s&pporte! b$ in or%ane""ofootprintin% e6perients )+52+9.0 Both a4bp spacin% 'one he"ica" t&rn/ bet(een the tTFA


3/40

bin!in% site an! the start site of transcription )+8.0The transcription factor has the abi"it$ to (rap an!&n(in! DNA in #itro in an essentia""$ non4specicprotein4DNA cop"e6 )+32++. an! scannin% transissione"ectron icroscop$ recent"$ re#ea"e! thatthe enop&s hoo"o%&e ca&ses sharp ben!in% of

the DNA !&p"e6 at the prooter acti#ation site)+.0 These tTFA4in!&ce! conforationa" chan%esof tDNA a$ be re1&ire! to a""o( the core NApo"$erase access to the tep"ate for initiation ofthe transcription process0DNase footprintin% an! transcription st&!ies ha#ein!icate! that the aGnit$ of tTFA for its bin!in%site ie!iate"$ &pstrea of the =SP is re"ati#e"$stron% an! that tTFA ar:e!"$ enhances =4stran!transcription0 Con#erse"$2 the aGnit$ for its bin!in%site &pstrea of the HSP is se#era" fo"! (ea:er an!tTFA on"$ o!erate"$ sti&"ates H4stran! transcription

)823923*2+5.0 This s&%%ests that a!!itiona"factors2 not operatin% in the in #itro s$ste a$ benecessar$ for e;ecti#e transcription initiation at theHSP in #i#o0 As aa"ian itochon!ria" NApo"$erase has not been p&rie! to hoo%eneit$2other accessor$ proteins a$ be present in the acti#eitochon!ria" NA po"$erase fractions0 A"tho&%hthere is no !irect e#i!ence for an$ aa"ian s&pp"eentar$coponents at the present tie2 an a!!itiona"8>4:Da

protein2 ob"i%ator$ for prooter!irecte!transcription se"ecti#it$ of the core itochon!ria"NA po"$erase2 has been i!entie! inse#era" $east species )+-,>. an! enop&s "ae#is)529.0 This factor2 !esi%nate! tTFB2 e6hibits se1&encehoo"o%$ to the !issociab"e c s&b&nit ofbacteria" NA po"$erases )+7. (hich is responsib"efor prooter reco%nition of the bacteria" ho"o4en$e0*090 E"on%ation an! terination of transcriptionOnce initiate! at the =SP2 the =4stran! is transcribe!as a sin%"e po"$cistronic prec&rsor NA2 encopassin%

ost2 if not a""2 %enetic inforation potentia""$enco!e! on the stran! )*28.0 A"tho&%h theHSP a$ !irect transcription of the entire H4stran!in a sii"ar fashion2 a ore cop"icate! o!e" hasbeen post&"ate! b$ Attar!i an! co""ea%&es )8>.0 Ine6ponentia""$ %ro(in% He=a ce""s2 the rNAs ares$nthesise! at a &ch hi%her rate than the in!i#i!&a"NAs enco!e! on the H4stran! )3.0 This !i;erencehas been e6p"aine! in part b$ the e6istence in#i#o of t(o !istinct initiation sites 'ITH5 an! ITH9 @Fi%0 9/ for H4stran! transcription )*7.0 Accor!in% tothe !&a" H4stran! transcription o!e"2 transcription

starts re"ati#e"$ fre1&ent at the ITH5 an! then terinatesat the !o(nstrea en! of the 5+S rNA %ene0This transcription process is responsib"e for s$nthesis


4/40

of the #ast a> bp apart can !eterine thefate of NA s$nthesis at the !ista" en! of the 5+SrNA %ene2 ore than 93>> n&c"eoti!es !o(nstrea0In a!!ition to the !&a" H4stran! transcriptiono!e"2 the !i;erence in s$nthesis rate of rNAan! NA has been e6p"aine! b$ an atten&atione#ent at the bor!er of the 5+S rNA an!

tNA=e&00 %enes 'Fi%0 5/0 The rst in!ication ofear"$ terination of the po"$cistronic H4stran! transcriptcae fro str&ct&ra" ana"$sis of the *P4en!s of5+S rNA o"ec&"es (hich re#ea"e! that a "ar%eproportion of the o"ec&"es ha#e ra%%e! *P4terinithat are co!e! for b$ the ie!iate"$ a!.0 The footprint encopassesa conser#e! tri!ecaer se1&ence b"oc: (ithin thetNA=e&00 %ene 'n&c"eoti!e positions *9*,*987of the h&an tDNA@ Fi%0 5/2 (hich has beensho(n b$ in #itro !e"etion &ta%enesis e6perientsto be essentia" an! s&Gcient for !irectin% terination)-5.0

The factor e!iatin% atten&ation of transcription

has been tere! TEF )->. or tTEM )-9. an!is :no(n to in!&ce ben!in% of the DNA he"i6 )-*.0 In#itro transcription st&!ies ha#e sho(n that tTEM


5/40

bo&n! to its tDNA tar%et site f&nctions bi!irectiona""$an! sho(s an e#en %reater eGcienc$ of terinationin the re#erse orientation re"ati#e to theprooter site )-8.0 Th&s2 in a!!ition to an atten&ationf&nction for H4stran! transcription2 tTEMa$ ha"t =4stran! transcription at a site (here no

=4stran! enco!e! %enes are fo&n! !o(nstrea 'seeFi%0 5/0 Bo&n! tTEM probab"$ stops e"on%ationof transcription b$ constit&tin% a ph$sica" barrier2rather than b$ a specic interaction (ith the itochon!ria"NA po"$erase2 beca&se tTEM a"soe!iates terination of transcription b$ hetero"o%o&sNA po"$erases )-*.0Both C"a$tons an! Attar!is %ro&ps ha#e sho(nthat a po"$pepti!e fraction of a itochon!ria" "$satecontainin% po"$pepti!es (ith a o"ec&"ar (ei%ht ofaro&n! *8 :Da has the abi"it$ to pro!&ce the specicDNase I footprint at the terination site associate!

(ith tTEM f&nction )-92-3.0 The cDNA of thepre!oinant po"$pepti!e fro this fraction (as recent"$c"one! an! se1&ence! )-+.0 The po"$pepti!econtains t(o (i!e"$ separate! basic re%ions an!three "e&cine ipper otifs (hich (ere sho(n to benecessar$ for its specic DNA4bin!in% capacit$ )-+.0

The footprint pro!&ce! b$ the recobinant protein(as sii"ar b&t not i!entica" to that pro!&ce! b$ the*84:Da po"$pepti!e fraction0 The recobinant protein(as a"so sho(n to be &nab"e to proote transcriptionterination in an in #itro s$ste )-+.0 Theseobser#ations s&%%est that an a!!itiona" coponent

of the *84:Da po"$pepti!e fraction is re1&ire! forthe terination acti#it$0 This apparent cop"e6it$of tTEM is not s&rprisin%2 %i#en that it sho&"!be ab"e to o!&"ate its acti#it$ in response to thece""&"ar !ean! for itochon!ria" rNAs2 on theone han!2 an! for itochon!ria" tNAs an!NAs on the other0Interestin%"$2 a heterop"asic A to ? transition inthe i!!"e of the tTEM bin!in% site 'A*98*?/ isfre1&ent"$ fo&n! in patients (ith the itochon!ria"encepha"o$opath$ ME=AS 'itochon!ria" $opath$2encepha"opath$2 "actic aci!osis an! stro:e4"i:e

episo!es/ )-. an! in patients (ith aterna""$ inherite!a!&"t onset !iabetes )--.0 In #itro st&!ies ha#esho(n that this &tation !raatica""$ re!&ces theaGnit$ of tTEM for its bin!in% site an! ca&sesa !efect in transcript terination )-92-*2-7.0 In contrast2stea!$4state transcript "e#e"s &pstrea an!!o(nstrea of the terination site (ere not a;ecte!b$ the A*98*? transition in c&"t&re! ce""s )-727>. orin tiss&es of patients )87275279.0 Th&s2 it sees &n"i:e"$that the in #itro obser#e! !efecti#e atten&ationof itochon!ria" transcription is of patho"o%ica" si%nicance0

*0*0 Processin% of priar$ transcriptsOnce theNA po"$erase passes the 5+S rNAJ


6/40

tNA=e&00 bo&n!ar$2 H4stran! transcription ap4

pears to be strai%htfor(ar!0 As no intron se1&encesare present in #ertebrate tDNA an! inter%eneticse1&ences are inia"2 processin% of the "on% po"$cistronicH4 an! =4stran! essen%ers is tho&%ht to

be a re"ati#e"$ sip"e process2 re1&irin% on"$ a fe(en$es0 ?enes for tNAs an: the t(o rNA%enes an! near"$ e#er$ protein %ene 'Fi%0 5/0 This&ni1&e %enetic or%anisation has "e! to the proposa"that the secon!ar$ str&ct&re of the tNA se1&encespro#i!e the p&nct&ation ar:s in the rea!in% of thetDNA inforation )9.0 Precise en!on&c"eo"$tic e6cisionof the tNAs fro the nascent transcripts (i""concoitant"$ $ie"! correct"$ processe! rNAs an!2in ost cases2 correct"$ processe! NAs )927*.0 Inthose cases in (hich the NA terini cannot beacco&nte! for b$ tNA e6cision 'e0%0 the essen%er

for s&b&nit I of c$tochroe c o6i!ase )927*.@ Fi%05/2 the processin% en$e possib"$ reco%nises a secon!ar$str&ct&re (hich shares critica" feat&res (iththe t$pica" c"o#er"eaf str&ct&res of tNAs0Mat&ration of itochon!ria" tNAs in#o"#esthree en$atic acti#ities (hich (ere recent"$ i!enti4e! b$ ossanith an! co""ea%&es )78. in an in #itroHe=a ce"" itochon!ria" tNA processin% s$ste0

Their e6perients sho(e! that c"ea#a%e at the3P4en! prece!es that at the *P4en!0 The en!on&c"easeresponsib"e for *P4en! c"ea#a%e has not been characterise!0C"ea#a%e at the 3P4en! is perfore! b$ a

itochon!ria" Nase P 'tNase P/0 The en$econtainin% fractions prepare! b$ ossanith an!co""ea%&es )78. c&t itochon!ria" tNA prec&rsorsat the correct 3P4en!2 b&t2 &n"i:e the preparationsb$ others 'see2 e0%0 )73./2 !o not c"ea#e tNAT$r prec&rsorsof Escherichia co"i correct"$0 This s&%%eststhat pre#io&s preparations (ere containate! (itha c$toso"ic isofor of Nase P (hich is apparent"$ab"e to acc&rate"$ process bacteria" tNA prec&rsors0

Yeast tNase P has been characterise! in !etai"0The en$e of Saccharo$ces cere#isiae is copose!of a n&c"ear4enco!e! protein an! a tDNAenco!e!

NA species )7+27.0 The NA oiet$ ofthe ribon&c"eoprotein cop"e6 is A4rich an! forsthe cata"$tic core of the en$e0 Coparison oftNase P NA fro !i;erent $east species hasre#ea"e! a rear:ab"e #ariation in sie fro 87> to58> n&c"eoti!es )7-.0Mat&ration of the e6cise! tNAs is cop"ete! b$a!!ition of the se1&ence CCA to their *P4en! cata"$se!b$ ATP'CTP/tNA n&c"eoti!$"transferase)78.0 Mitochon!ria" NAs are po"$a!en$"ate! b$a itochon!ria" po"$'A/ po"$erase !&rin% or ie!iate"$after c"ea#a%e )7725>>.2 (hereas the *P4en!s of

the t(o rNAs are post4transcriptiona""$ o!ie!b$ the a!!ition of on"$ short a!en$" stretches )-.0Mitochon!ria" essen%ers !o not carr$ &pstrea


7/40

po"$a!en$"ation si%na"s as fo&n! in n&c"ear essen%ers080 Mitochon!ria" protein s$nthesis8050 Mitochon!ria" ribosoesEar"$ in!ications of the pro:ar$otic ori%in of itochon!riacae fro obser#ations2 no( ore thanthree !eca!es a%o2 that the spectr& of antibiotics

inhibitin% itochon!ria" protein s$nthesis reseb"esthat of pro:ar$otic s$stes 're#ie(e! in )5>5./0 S&bse1&entresearch conre! that near"$ a"" constit&entsof the itochon!ria" trans"ation achiner$ are!istinct fro their c$toso"ic co&nterparts0 ni1&efeat&res of itochon!ria" protein s$nthesis (ere rstst&!ie! at the o"ec&"ar "e#e" in itochon!ria" ribosoes0

The itochon!ria" ribosoes2 or itoribosoes2are "ocate! in the atri6 of the or%ane""e0Stea!$4state rNA "e#e"s in rat hepatoc$tes )5>9.s&%%est that there are +5>> itoribosoes per itochon!rion0

The ph$sica" an! cheica" properties of

itoribosoes !i;er consi!erab"$ fro their c$toso"icas (e"" as their bacteria" co&nterparts0 Maa"ianitoribosoes ha#e an &n&s&a""$ "o( NA contentan!2 conse1&ent"$2 a "o( se!ientationcoeGcient of L33S )5>*,5>+.0 The L*7S an!L9-S ribosoa" s&b&nits contain respecti#e"$ the5+S an! 59S rNA species enco!e! b$ the tDNA)5>*25>8.0 A 3S rNA species2 t$pica""$ present inribosoes2 appears to be absent in aa"ian itoribosoes)5>*25>8. b&t a 9*4bp re%ion at the*P4en! of the h&an 5+S rNA e6hibits a +- se1&encei!entit$ to a portion of the Baci""&s s&bti"is 3S

rNA0 The str&ct&ra" sii"arit$ of the re%ion an!its appin% position ha#e "e! to the s&%%estionthat this piece represents a tr&ncate! 3S rNA(hich reaine! part of the "ar%e rNA coponent)5>.0

The "o( NA content of aa"ian itoribo4

soes is copensate! b$ a re"ati#e"$ hi%h proteincontent an! res&"ts in a tota" ass of itoribosoessii"ar to that of bacteria" ribosoes0 T(o4!iensiona"%e" e"ectrophoresis has a""o(e! the reso"&tionof -3 itoribosoa" protein spots fro beef )5>-.

an! -+ fro rat )5>+.2 ho(e#er2 the act&a" n&berof !istinct itoribosoa" proteins a$ be "o(er as!i;erences in stainin% intensit$ of soe spots s&%%estpossib"e proteo"$tic !e%ra!ation an! containation(ith other proteins cannot f&""$ be e6c"&!e!0 Onecan spec&"ate that soe itoribosoa" proteinsha#e a!opte! str&ct&ra" an! f&nctiona" ro"es of the"ost rNA b&t e6perienta" !ata as to the f&nctionsof aa"ian itoribosoa" proteins are nota#ai"ab"e an! ost recent pro%ress on the e"&ci!ationof the properties of the in!i#i!&a" itoribosoa"proteins coes fro st&!ies in S0 cere#isiae )5>7.0

8090 Initiation an! e"on%ation of trans"ationA"tho&%h iso"ate! intact itochon!ria faithf&""$carr$ o&t protein s$nthesis2 an in #itro itochon!ria"


8/40

trans"ation s$ste &sin% on"$ itochon!ria" e6tractsis not a#ai"ab"e0 D&e to this persistent "ac:2 an$!etai"s of the itochon!ria" protein bios$nthesis arepoor"$ &n!erstoo! an! on"$ a "iite! n&ber of theaa"ian a&6i"iar$ factors in#o"#e! in initiationan! e"on%ation of trans"ation ha#e been characterise!0

Terination of the trans"ation process has not$et been e6p"ore!0

The itochon!ria" trans"ationa" apparat&s is&ni1&e in an$ (a$s0 As entione! ear"ier2 therNA an! tNA species are s&rprisin%"$ sa""0

The start of the trans"ation process is intri%&in% beca&seaa"ian itochon!ria" NAs ha#e no&pstrea "ea!er se1&ences to faci"itate ribosoebin!in%2 &n"i:e pro:ar$otic an! e&:ar$otic c$toso"icessen%ers2 b&t start at or #er$ near the 3P4en! (iththe co!on for the initiatin% N4for$"ethionine )7*.0In a!!ition2 the 3P4terini of itochon!ria" NAs

"ac: a 4eth$"%&an$"ate cap str&ct&re )55>.0 Thise6c"&!es a cap reco%nition an! scannin% echanisfor !irectin% the ribosoe to the initiation co!on asobser#e! in the c$toso" of e&:ar$otic ce""s0 The "o(trans"ationa" eGcienc$ of itochon!ria" essen%ers)5>9. a$ in fact be the res&"t of the absence of a3P4en! ribosoe reco%nition site an! necessitate theobser#e! ab&n!ance of itochon!ria" essen%ers)5552559. to ens&re that a s&Gcient "e#e" of trans"ationocc&rs0In #itro e6perients (ith bo#ine itoribosoesha#e in!icate! that the sa"" '9-S/ ribosoa" s&b&nit

has the abi"it$ to bin! NA ti%ht"$ in a se1&encein!epen!entanner an! in the apparent absence ofa&6i"iar$ initiation factors or initiator tNA )55*.2&n"i:e pro:ar$otic )558. an! e&:ar$otic c$toso"ic)553. s$stes0 &!%in% fro the sie of the NAfra%ents protecte! fro Nase T5 !i%estion2 thea,->4n&c"eoti!e stretch2b&t L8>> n&c"eoti!es are inia""$ re1&ire! for ef4cient bin!in% )55*255+255.0 This a$ e6p"ain (h$the t(o shortest e6presse! open rea!in% fraes ofaa"ian tDNA 'ATPase- an! ND8=@ +*>>

bp/ are both part of o#er"appin% %enes 'ATPase-JATPase+ an! ND8=JND8@ see Fi%0 5/0 Both pairsof %enes res&"t in !icistronic essen%ers )5>>.0 Monocistronictranscripts of the ATPase- an! ND8= %enesare possib"$ too short to interact e;ecti#e"$ (ith thesa"" s&b&nit0After bin!in% of the sa"" ribosoa" s&b&nit tothe essen%er2 the s&b&nit is ass&e! to o#e tothe 3P4en! of the NA e!iate! b$ $et &nspecie!a&6i"iar$ initiation factors )55+255-.0 The on"$ initiationfactor i!entie! in aa"ian itochon!ria to!ate is tIF49 )55-2557.0 The cDNAs for bo#ine as

(e"" as h&an tIF49 ha#e been c"one! an! se1&ence!)59>2595.0 The h&an factor sho(s *+


9/40

aino aci! i!entit$ (ith E0 co"i IF490 This onoericprotein factor be"on%s to the fai"$ of?TPases an! prootes fMet4tNA bin!in% to thesa"" ribosoa" s&b&nit in the presence of ?TPan! a tep"ate2 reiniscent of the bacteria" factorIF490 Detai"e! in #itro characterisation of bo#ine

tIF49 )5572599. has in!icate! that tIF49 a$bin! to the sa"" ribosoa" s&b&nit prior to its interaction(ith ?TP2 ho(e#er2 ?TP enhances the af4nit$ bet(een tIF49 an! the sa"" s&b&nit an! a""o(sfMet4tNA to .0 Di;erent fro E0co"i EF4T& an! EF4Ts2 aa"ian tEF4T& an!tEF4Ts for a ti%ht"$ associate! cop"e6 that2 &n"i:ethe bacteria" cop"e62 cannot rea!i"$ be !issociate!b$ %&ani!ine n&c"eoti!es a"one )59*.0 Ho(e#er2it (as recent"$ !eonstrate! that the tEF4T&4Tscop"e6 (i"" !issociate in the presence of ?TP an!char%e! tNAs )5*5.080*0 The e;ect of tNA point &tations on tDNAe6pressionPatho%enic &tations in itochon!ria" %enes are%enera""$ heterop"asic2 i0e0 (i"!4t$pe an! &tant

tDNA co4e6ist in the sae ce""0 The (i"!4t$petDNA a""o(s the &tant tDNA (ith the other(ise"etha" base chan%e to persist0 Materna""$ inherite!2itochon!ria" encepha"o$opathies are oftenassociate! (ith point &tations in itochon!ria"tNA %enes )5*9.0 M&sc"e biopsies fro patientssho( foca" histocheica" !efects of c$tochroe co6i!ase associate! (ith a non4ran!o !istrib&tionof &tant an! (i"!4t$pe tDNA )87275.0 This osaice6pression of the !isease is a"so obser#e! in priar$ce"" c&"t&res of these patients as i""&strate! inFi%0 *0

ecent pro%ress on ho( itochon!ria" tNA &tationse6ert their e;ect has "ar%e"$ coe fro e6perientsin (hich patients tDNA has been transferre!


10/40

to b> contro" ce""s0 These st&!ies &ne1&i#oca""$!eonstrate! that tNA point &tations2 "i:e theA*98*? transition fo&n! in the tNA=e&00 %eneof ME=AS patients an! the A-*88? transition inthe tNA=$s %ene coon"$ associate! (ithMEF '$oc"onic epi"eps$ (ith ra%%e!4re! bres/

)5*8.2 are a"one s&Gcient to ipair intraitochon!ria"protein s$nthesis )-727>25*3.0 These an! otherce"" c&"t&re st&!ies )5*+. ha#e re#ea"e! a steep thresho"!e;ect itochon!ria" trans"ation reains &naecte!b$ tNA point &tations &nti" s-3 ofthe tDNA o"ec&"es are &tate!0Di;erent echaniss can acco&nt for the obser#e!ipairent of protein s$nthesis0 Apart froa potentia" e;ect on transcription terination2 thetNA=e&00 A*98*? transition appears to a;ectthe processin% of priar$ itochon!ria" transcripts)7>279.0 C$bri!s res&"tin% fro re4pop&"ation e6perients

of a h&an b> ce"" "ine (ith tDNA carr$in%the A*98*? transition sho(e! a sa"" '"ess than 94fo"!/ b&t consistent increase in the stea!$4state "e#e"sof a partia""$ processe! NA species !eri#e! fro

transcription of the 5+S rNAtNA=e&00ND5%enes )7>25*.2 (hich are conti%&o&s in the tDNA'Fi%0 5/0 Stea!$4state "e#e"s of at&re 5+S rNA2tNA=e&00 an! ND5 NA are not a;ecte! b$the &tation )7>25*.0 A"tho&%h the stea!$4state "e#e"of the iat&re transcript is e6tree"$ "o( in thec$bri!s copare! to the "e#e" of at&re 5+S

rNA2 the "e#e" sho(e! a stron% in#erse corre"ation(ith the rates of o6$%en cons&ption of the c$bri!s'an in!icator of itochon!ria" o6i!ati#e phosphor$"ationcapacit$ )5*-25*7./0 These obser#ations ha#e"e! to the h$pothesis )7>25*-. that the &nprocesse!transcript2 (hich contains 5+S rNA2 is incorporate!into ribosoes ren!erin% the f&nctiona""$ !e4cient0 If this res&"ts in sta""in% of the trans"ation ofpo"$ribosoa" NAs2 then a sa"" increase of &nprocesse!transcript co&"! interfere !isproportiona""$(ith itochon!ria" trans"ation an! e6p"ain the se#ereo6i!ati#e phosphor$"ation !efects obser#e! in the

patients0A"ternati#e"$2 point &tations in tNA %enes a$in!&ce a conforationa" chan%e of the tNA res&"tin%in a !ecrease! stabi"it$ of the o"ec&"e0 A chan%ein str&ct&re a$ a"so a;ect the i!entication b$ theco%nate ainoac$"4tNA s$nthase an! "ea! to !ecrease!"e#e"s of ainoac$"ation or e#en ischar%in%of &tate! tNAs0 In pro:ar$otes2 the T-C re%ionof the tNA o"ec&"e is iportant for reco%nitionan! bin!in% b$ EF4T& an! ribosoes2 therefore2 &tationsin the T-C re%ion of itochon!ria" tNAsare "i:e"$ to a;ect their aGnit$ (ith tEF4T& an!

itoribosoes0 Hi%h reso"&tion Northern 'NA/b"ot h$bri!isation e6perients ha#e in!icate! thatthese echaniss are in!ee! "i:e"$ to p"a$ a ro"e in


11/40

the aetio"o%$ of these !iseases0 In ce"" "ines carr$in%either tNA=$s A-*88? )58>2585.2 tNA=e&00C*93+T )589.2 or tNAAsn ?3>*A &tations)58*.2 ar:e!"$ re!&ce! stea!$4state "e#e"s of the aecte!tNA ha#e been fo&n!2 s&%%estin% an increase!s&sceptibi"it$ to n&c"eo"$tic !i%estion0 Moreo#er2

a L8> re!&ction in ainoac$"ation oftNA=$s has been !eonstrate! in ce"" "ines (iththe tNA=$s A-*88? &tation )585.0 In contrast2ce"" "ines (ith the tNA=$s C-*3+T &tation e6hibite!no si%nicant !ecrease in tNA=$s content )58>.b&t this &tation2 (hich is "ocate! in the T-C arof the o"ec&"e2 a$ interfere (ith the bin!in% oftEF4T& or the itoribosoe )588.030 ep"ication of tDNA3050 Basic echanis of aa"ian tDNArep"ication

The fort&ito&s"$ s"o( rate of tDNA rep"ication

has faci"itate! the iso"ation an! characterisation of in#i#o rep"icati#e intere!iates an! has pro#i!e! theno( %enera""$ accepte! o!e" of the rep"ication c$c"eof aa"ian tDNA0 Ear"$ st&!ies2 (hich pre!oinant"$re"ie! on e"ectron icroscopic an! centrif&%a"ana"$sis of tDNA o"ec&"es fro c&"t&re!ce""s2 in!icate! that aa"ian tDNA o"ec&"esrep"icate! &ni!irectiona""$ fro t(o spatia""$ an!tepora""$ !istinct2 stran!4specic ori%ins )9*.0 Theori%in of H4stran! rep"ication 'OH/ is "ocate! !o(nstreaof the =SP in the D4"oop re%ion of the %enoe2(hereas the ori%in of =4stran! rep"ication

'O=/ is at t(o4thir!s of the %enoic !istance a(a$fro OH (ith respect to the po"arit$ of H4stran!s$nthesis 'Fi%0 5/0 A ro&n! of rep"ication be%ins atOH (ith the s$nthesis of a !a&%hter H4stran! an!contin&es a"on% the parenta" =4stran! to pro!&ce af&"" H4stran! circ"e0 On"$ after the rep"ication for:has passe! the secon! rep"ication ori%in2 O=2 is s$nthesisof the =4stran! initiate! (hich procee!s in a!irection opposite to that of H4stran! rep"ication're#ie(e! in )583./03090 Initiation of H4stran! s$nthesisFine appin% of NA an! DNA species in the D"oop

re%ion of h&an an! o&se tDNA ha#e s&%%este!that short itochon!ria" transcripts2 ori%inatin%at IT=2 ser#e as priers for the initiation of s$nthesisof nascent H4stran!s 'Fi%0 9@ )58+258./0 Th&s2rep"ication of aa"ian tDNA appears to be intiate"$"in:e! (ith itochon!ria" transcription0

There are no :no(n !i;erences bet(een the initiationof =4stran! transcription an! the initiation ofNA prier foration for tDNA rep"ication )*+.an! it is not c"ear (hich echanis !eci!es bet(eentranscript e"on%ation or H4stran! s$nthesis0 Transitionsfro NA to DNA s$nthesis ta:e p"ace at

se#era" !istinct sites that co""ecti#e"$ constit&te OHin a re%ion of three short2 e#o"&tionar$ conser#e!


12/40

se1&ence b"oc:s2 nae! CSB I2 II an! III 'Fi%0 9@

As the prec&rsor NA prier e6ten!s be$on! thetransition sites of NA to DNA s$nthesis2 the priar$transcript is be"ie#e! to be en$atica""$ processe!to $ie"! the at&re prier NA *P4terini0

Beca&se of their "ocation2 it has been spec&"ate!that CSB I2 II an! III !irect the precise c"ea#a%e ofpriar$ transcripts to pro#i!e the appropriate prierspecies )*+.0 ecent in #itro transcription st&!iesof the OH re%ion (ith itochon!ria" NA po"$erasefractions )58-. in!icate! that the prec&rsor NAprier e6ists as a stab"e an! persistent NA4DNAh$bri! a"so :no(n as an 4"oop0 H$bri! forationre1&ires the CSB II e"eent an! is a"so a;ecte! b$&tations in CSB III0

The search b$ C"a$tons %ro&p for cata"$tic acti#it$capab"e of processin% =4stran! transcripts containin%

OH se1&ences "e! to i!entication of the en$eca""e! itochon!ria" NA processin%en!on&c"ease 'Nase MP@ re#ie(e! in )587./0 Intheir initia" st&!ies2 in (hich sin%"e4stran!e! OH4containin%NA species (ere &se! as s&bstrate2 the in#itro Nase MP c"ea#a%e sites !i! not atch (itha"" in #i#o 3P4en!s of the nascent h&an an! o&seH4stran!s 'cf0 )58+258253>2535./0 Ho(e#er2 recent"$=ee an! C"a$ton )539. !eonstrate! that o&seNase MP !oes c"ea#e the prec&rsor NA in theconte6t of a trip"e4stran!e! 4"oop con%&ration in#itro at the a.0Another n&c"ease ip"icate! in processin% of prec&rsor

NA priers for H4stran! rep"ication is en!on&c"ease? )5+5.0 The en$e (as rst iso"ate!fro bo#ine heart itochon!ria as a hoo!ier of


13/40

a L97 :Da po"$pepti!e )5+9. an! cDNA se1&encesspecif$in% en!on&c"ease ? of se#era" aa"ianspecies2 inc"&!in% h&an2 (ere recent"$ reporte!)5+*.0 The itochon!ria" "ocation of en!on&c"ease? is &n!isp&te! b&t the en$e is a"so fo&n! inthe n&c"e&s )5+525+8.0 En!on&c"ease ? has a rather

(i!e spectr& of n&c"eo"$tic acti#ities it c"ea#es?C4rich !o&b"e4stran!e! an! sin%"e4stran!e! DNAtracts2 NA an! an NA4DNA hetero!&p"e6 containin%the o&se OH )5+5.0 The in #itro NA c"ea#a%esites of the hetero!&p"e62 ho(e#er2 !o not a"i%n(ith a"" pre!icte! in #i#o priin% sites0 De"etion ofthe hoo"o%o&s %ene in S0 cere#isiae !oes not seeto a;ect tDNA etabo"is )5+3.0 Th&s2 an$ conc"&sionas to the f&nction of en!on&c"ease ? in itochon!ria"bio%enesis appears preat&re0In #ertebrates2 ost H4stran! s$nthesis e#ents sta""short"$ after initiation0 Arreste! nascent H4stran!s

reain annea"e! to their tep"ate =4stran! an! createthe trip"e6 D4"oop str&ct&re )583.0 The *P4en!s ofpreat&re"$ terinate! H4stran!s ap L3> n&c"eoti!es!o(nstrea of a short '53 bp/ conser#e! se1&encee"eent2 ca""e! the terination4associate! se1&ence'TAS )587./0 The n&ber of TAS e"eentsan! H4stran! terination sites #ar$ per species0

The h&an itochon!ria" %enoe contains on"$ asin%"e TAS e"eent an! there is on"$ one a n&c"eoti!esan! is an:e! b$ #e tNA %enes 'Fi%0 5/0 O=is on"$ acti#ate! (hen the parenta" H4stran! is !isp"ace!as a sin%"e stran! b$ the %ro(in% !a&%hter Hstran!0After stran! !isp"aceent2 O= is tho&%ht toa!opt a !istincti#e ste4"oop str&ct&re )*+.0 In #itro

r&n4o; rep"ication st&!ies of O=

ha#e s&%%este! thatthis con%&ration ser#es as the reco%nition str&ct&refor a itochon!ria" DNA priase (hich pro#i!es a


14/40

short NA prier for =4stran! s$nthesis )55259.0Ho(e#er2 the "ocation of O= (ithin a c"&ster oftNA %enes an! the fact that the potentia" ste"oopstr&ct&re i%ht be absent in tDNA of soe#ertebrate species )587. s&%%est that a!!itiona" secon!ar$str&ct&res contrib&te to DNA priase

reco%nition in #i#o0NA priin% starts at the T4rich portion of thepre!icte! O=4"oop an! the transition fro NA toDNA s$nthesis ta:es p"ace at a specic site near acritica" ?C4rich e"eent at the base of the hairpin)55259.0 The itochon!ria" DNA priase in#o"#e!in priin% of the =4stran! s$nthesis has on"$ beenpart"$ p&rie! )5*.0 The ph$sica" properties of theitochon!ria" en$e are !istinct fro the a


15/40

!oain0 ecent :inetic st&!ies (ith the recobinant"$e6presse!2 58>4:Da h&an s&b&nit sho(e!that the a*,59* 553

fro the n&c"ear sin%"e4stran!e! bin!in% protein b&treseb"es the E0 co"i sin%"e4stran!e! bin!in% proteinin str&ct&re as (e"" as in DNA4bin!in% properties)5-25--.0 The protein interacts (ith sin%"e4stran!e!DNA as a hootetraer )5-,5-7.0 The cr$sta"str&ct&re of h&an tSSB s&%%ests that sin%"estran!e!DNA (raps aro&n! the tetraeric cop"e6thro&%h e"ectropositi#e channe"s %&i!e! b$ e6ib"e

"oops )5-7.0Se#era" a!!itiona" en$atic acti#ities are consi!ere!to be essentia" for tDNA rep"ication b&t on"$a fe( s&pp"eentar$ en$es of the itochon!ria"rep"ication achiner$ ha#e been i!entie!0 He"icasescata"$se the &n(in!in% of !&p"e6 DNA b$ !isr&ptin%the h$!ro%en bon!s that ho"! the t(o stran!s to%etherto pro#i!e sin%"e4stran!e! tep"ates for DNA po"$erases)57>.0 An ATP4!epen!ent he"icase has beeni!entie! an! part"$ p&rie! fro itochon!ria ofbo#ine brain )575. an! sea &rchin e%%s )579.0 Theen$e sho(s biocheica" sii"arities (ith ep he"icase

of E0 co"i an! shares its *PC3P po"arit$ of &n(in!in%(ith respect to the sin%"e4stran!e! portionof the partia" !&p"e6 DNA0 This !irection (o&"!p"ace the itochon!ria" he"icase on the tep"atestran! ahea! of DNA po"$erase Q !&rin% tDNArep"ication0

T$pe I topoisoerases cata"$se the re"a6ation ofs&percoi"e! DNA2 (hi"e t$pe II topoisoerases areresponsib"e for the intro!&ction of s&percoi"s0 Topoisoeraseschan%e the topo"o%$ of DNA b$ transient"$brea:in% the bac:bone bon!s2 &sin% a echanisin#o"#in% either sin%"e stran! 't$pe I/ or !o&b"e

stran! 't$pe II/ c"ea#a%e )57*.0 Mitochon!ria""$ associate!2t$pe I topoisoerases ha#e been i!entie!fro #ario&s so&rces2 inc"&!in% h&an "e&:eia


16/40

ce""s )578. an! h&an p"ate"ets )573.0 The itochon!ria"en$e is i&no"o%ica""$ re"ate! to its n&c"earco&nterpart )57+257. an! $east itochon!ria""$ associate!t$pe I topoisoerase acti#it$ is abo"ishe!(hen the %ene that enco!es the n&c"ear t$pe I topoisoeraseis !isr&pte! )57-.0 Despite the apparent

i&no"o%ica" an! %enetic re"ationship bet(een then&c"ear an! itochon!ria" t$pe I topoisoerases2 theen$es can be !istin%&ishe! fro each another b$ a!i;erentia" response to cheica" rea%ents an! inhibitors2both in $east an! h&ans )573257.0Mitochon!ria""$ associate! t$pe II topoisoerasesha#e been i!entie! in Dict$oste"i& !iscoi!e&)577.2 rat "i#er )9>>. an! h&an "e&:eia ce""s)578.0 F&rther s&pport for the e6istence of a !istinctitochon!ria" t$pe II topoisoerase coes fron!in%s (ith ce"" c&"t&res %ro(n in the presence ofciproo6acin0 This antibiotic inhibits pro:ar$otic

t$pe II topoisoerases &ch ore than the e&:ar$oticn&c"ear en$e0 Treatent of c&"t&re! ce""s (ithciproo6acin ca&ses !o&b"e4stran!e! brea:s intDNA an! res&"ts in "oss of the itochon!ria" %enoe)9>5.0 These obser#ations s&%%est the presenceof a bacteria"4"i:e t$pe II topoisoerase in itochon!ria03030 A ro"e for tTFA in tDNA aintenanceIn a!!ition to its ro"e in transcription an!2 conse1&ent"$2prier foration for H4stran! s$nthesis2tTFA appears to ha#e a f&nction in aintenanceof tDNA0 Mitochon!ria !o not contain histones)9>929>*.0 Yeast tTFA )9>8. contains a tan!e of

HM? DNA4bin!in% otifs2 sii"ar to tTFA of#ertebrates2 b&t $east tTFA "ac:s the basic C4terina"re%ion sho(n to be iportant for prooterse"ection in #ertebrates )+>.0 The $east hoo"o%&eis not consi!ere! to p"a$ a :e$ ro"e in initiation oftranscription )*. b&t is tho&%ht to ha#e a histone"i:ef&nction in coatin% the entire itochon!ria" %enoe)++29>9.0 =i:e(ise2 bin!in% of #ertebratetTFA is not restricte! to the re%ions &pstrea ofthe HSP an! =SP0 The factor is inherent"$ e6ib"e inits reco%nition of DNA se1&ences )392+325. an! acoparati#e in or%ane""o an! in #itro footprintin%

st&!$ in!icate! that h&an tTFA bin!s at re%&"ar"$space! inter#a"s thro&%ho&t a 3>>4bp re%ionencopassin% OH an! the t(o a


17/40

tTFA :noc:o&t ice e6hibit re!&ce! tDNAcop$ n&bers an! hoo$%o&s :noc:o&t ebr$os55+ 04W0 Taanan JBiochiica et Bioph$sica Acta 585> '5777/ 5>*,59*

"ac: tDNA an! !ie )9>3.0 This !eonstrates theiportance of tTFA in aintainin% tDNA "e#e"sin #i#o0 Con#erse"$2 in b> ce"" c&"t&res tTFA "e#e"s

are "o( )9>+,9>-.2 (hi"e in patients (ith itochon!ria"$opathies increase! "e#e"s of tTFA arefo&n! in ra%%e!4re! &sc"e bres (ith acc&&"ate!"e#e"s of tDNA )9>+.0 A"tho&%h tTFA protein"e#e"s are "o( in b> ce""s2 tTFA NA "e#e"s arenora" )9>+29>.2 in!icatin% that tTFA protein "e#e"sare post4transcriptiona""$ re%&"ate! b$ tDNA"e#e"s0 Possib"$2 bin!in% to tDNA protects tTFAfro proteo"$tic !e%ra!ation0 Sii"ar"$2 tSSB protein"e#e"s appear to corre"ate (ith tDNA contentb&t the e6pression of tSSB is at "east in part re%&"ate!at the "e#e" of transcription )9>7.0 On the other

han!2 DNA po"$erase Q is constit&ti#e"$ e6presse!)9>7.@ e#en in b> ce""s2 the stea!$4state "e#e" of thepo"$erase protein is &na;ecte! )9>-.0Interestin%"$2 the e6pression of tTFA is re!&ce!!&rin% aa"ian sperato%enesis2 ost "i:e"$ !&eto the pro!&ction of a"ternate2 testis4specic tTFAtranscript isofors )95>2955.0 The "o( tDNA cop$n&ber obser#e! in sper ce""s a$2 therefore2 be a!irect res&"t of a !ecrease! e6pression of tTFA(hich2 to%ether (ith other echaniss2 a$ pre#entpaterna" transission of tDNA )955.0+0 Prospects

Transcription2 trans"ation an! rep"ication oftDNA are contro""e! b$ an$ factors0 Cis4actin%tDNA se1&ences ha#e been i!entie! in appin%st&!ies an! b$ &tation ana"$sis0 Characterisation oftrans4actin% factors pro#e! ore !iGc&"t beca&se oftheir "o( ce""&"ar concentration an! the potentia" ris:of containation (ith ana"o%o&s en$e acti#ities ofn&c"ear or c$toso"ic ori%in in ce"" fractionation e6perients0Sti""2 soe re"ati#e"$ ab&n!ant itochon!ria"en$es co&"! be p&rie! to hoo%eneit$ an!their h&an co!in% se1&ences ha#e been c"one! &ti"isin%con#entiona" o"ec&"ar bio"o%ica" techni1&es0

More recent"$2 prob"es (ith p&rication ha#ebeen circente! b$ screenin% of h&an EST !atabases(ith se1&ences of $east %enes in#o"#e! in transcriptionan! rep"ication )35.0 As a res&"t2 se#era" :e$en$es in#o"#e! in transcription an! rep"ication ofh&an tDNA ha#e no( been c"one! an! appe!to chroosoes 'Tab"e 5/0 Ne#erthe"ess2 cop"eentar$strate%ies are nee!e! to characterise thef&"" repertoire of en$es in#o"#e! in tDNA e6pressionas e#o"&tionar$ !i#er%ence bet(een factorsa$ pre#ent s&ccessf&" c$berscreenin% an! certainfactors co&"! be &ni1&e to the aa"ian itochon!ria"

s$ste0Patients (ith inborn errors of itochon!ria" rep"ication2transcription or protein s$nthesis present a


18/40

cha""en%e as (e"" as a reso&rce to the biocheist0 Thecha""en%e is to &n!erstan! the o"ec&"ar echanisof the !isease2 (hereas the reso&rce is as a nat&ra""$occ&rrin% &tant (hich pro#i!es an opport&nit$ tocharacterise aspects of these processes in h&ansthat are !iGc&"t to st&!$ b$ other eans0 Patients

(ith tDNA &tations are pro#i!in% &npara""e"e!insi%hts into the ro"es of cis4actin% e"eents0 Withrespect to trans4actin% factors in#o"#e! in aintenancean! rep"ication of tDNA2 one %ro&p of patientshas attracte! specia" attention0 In 57752 Moraesan! co""ea%&es )95+. !escribe! se#era" infants(ith ar:e! !ep"etion of tDNA an! !efecti#e o6i!ati#ephosphor$"ation0 Since its !isco#er$2 orethan *> patients ha#e been !escribe! (ith this s$n!roe'!isc&sse! in )95./0 Most patients presentsoon after birth (ith &sc"e (ea:ness an! hepaticfai"&re or rena" t&b&"opath$ associate! (ith a se#ere

!ep"etion of tDNA '--,77/ in a;ecte! tiss&es at

post4orte0 No tDNA &tations ha#e been!oc&ente! in these infants an! pe!i%ree ana"$sisis consistent (ith a&tosoa" inheritance of the trait0A n&c"ear %enoic in#o"#eent has been conre!in t(o fai"ies b$ tDNA transfer techni1&es)95-25**.0 The pro%ressi#e "oss of tDNA in tiss&esis a"so obser#e! in ce"" c&"t&res of soe of the patients)95-25**.0 In the !ep"etin% c&"t&res2 a fractionof the ce""s sti"" contains apparent"$ nora" "e#e"s oftDNA b&t tDNA rep"ication is ore or "ess !iinishe!

in a"" ce""s 'Fi%0 8/0 Ta:en to%ether2 theseobser#ations stron%"$ s&%%est a rep"ication arrest !&eto a !ecienc$ of a trans4actin%2 n&c"ear4enco!e! factor0Patients (ith tDNA !ep"etion !isp"a$ !ecrease!"e#e"s of tTFA in tiss&e )9>+29>.0 Ho(e#er2 thefact that renant tDNA is sti"" transcribe! in ce""c&"t&res of the patients )5**2957. s&%%ests that the!ecrease in tTFA "e#e"s is probab"$ secon!ar$ to!ep"etion of tDNA0 Other can!i!ate %enes2 "iste!in Tab"e 52 are c&rrent"$ bein% e#a"&ate! b&t2 to !ate2no o"ec&"ar !efects &n!er"$in% the tDNA !ep"etionha#e been reporte!0 A !ecienc$ of one of the

:e$ en$es of tDNA rep"ication is "i:e"$ to be"etha" ear"$ in ebr$onic !e#e"opent )9>3.0 The apparenttiss&e4specic an! neonata" e6pression ofs$ptos in!icates a "ess !rastic !efect an! pointsto the !ecienc$ of a factor in#o"#e! in re%&"ation oftDNA cop$ n&bers0 e%&"ator$ factors are %enera""$present at #er$ "o( ce""&"ar concentrations an!on"$ at certain sta%es of !e#e"opent2 a:in% it e6cee!in%"$!iGc&"t to characterise these factors b$ tra4

!itiona" eans0 It a$2 ho(e#er2 be feasib"e to i!entif$the !ecient factor in cop"eentation st&!ies of

tDNA !ep"etin% ce"" c&"t&res (ith a h&an cDNAe6pression "ibrar$0 This approach is faci"itate! b$ thefact that the patients ce""s2 beca&se of ipairent of


19/40

the o6i!ati#e phosphor$"ation cop"e6es2 becoea&6otrophic for &ri!ine an! p$rate )95-25**.2(hich pro#i!es se"ectab"e ar:ers to i!entif$ cop"eentin%cDNAs0One can anticipate that f&t&re pro%ress in the e"!of rep"ication2 transcription an! trans"ation of h&an

tDNA (i"" increasin%"$ coe fro !etai"e!case st&!ies as e6ep"ie! abo#e for tDNA !ep"etion0

This research (i"" not on"$ enrich science b&t(i"" a"so perit the !e#e"opent of !ia%nostic too"sas (e"" as ne( patho%enic insi%hts to iniise the!e#astatin% conse1&ences of a !efecti#e e6pression ofthe itochon!ria" %enoe0


20/40

LapakTranslate

Inisiasi transkripsi

Mekanisme dasar transkripsi mitokondria

telah diselesaikan pada spesies wakil dari beberapa

kelompok filogenetik (Ulasan di [36 ^ 38]! Man"sia

sit"s inisiasi transkripsi dan mt#$%

daerah promotor telah ditent"kan dengan mengg"nakan &arietas

teknik' termas"k pemetaan )*akhir primer

transkrip mitokondria oleh perlind"ngan n"klease +,

eksperimen [3- ^ .,] dan penghap"san [./ ^ ..]' sitedire0ted

m"tagenesis [.] dan linker s"bstit"si

analisis [.6] fragmen mt#$% kloning di 1"no

2Tes transkripsi! +em"a data ang tersedia adalah

konsisten dengan kesimp"lan bahwa ada dua

situs inisiasi transkripsi utama dalam D-lingkaran

(ITH1 dan ITL) terletak dalam 150 bp dari satu sama lain(Gambar. 2). Unsur prmtr dengan knsensus 15-bp

mti! urutan" 5#-$%&%$$ (G) $$ (%) %%%G%'%"

mengelilingi situs inisiasi transkripsi (digarisbaai)

dan sangat penting untuk transkripsi *+,"+5.

H-untai transkripsi dimulai pada psisi nukletida

51 (ITH1/ penmran menurut *21) terletak

dalam prmtr H-strand (H#) dan segera

berdekatan dengan gen t&%#e" sedangkan

L-untai transkripsi dimulai pada psisi nukletida

+0 (ITL) 3ang terletak di dalam prmtr L-strand

(L#). 4lemen penamba ulu tambaandiperlukan untuk transkripsi 3ang ptimal(4ambar! /! Uns"r*"ns"r ini'
https://translate.google.com/https://translate.google.com/


21/40

ang kem"dian ditampilkan "nt"k menertakan mengikat

sit"s "nt"k faktor transkripsi (mtT5%' lihat di bawah'

"r"t pameran kesamaan tetapi hana ika salah sat" "ns"r adalah

terbalik relatif terhadap lainna [.3'.'.7]!

Meskip"n dekat dari +) dan L+)'

awal dalam st"di transkripsi &itro men"n"kkanbahwa "ns"r*"ns"r terseb"t se0ara f"ngsional independen

[./'.3'.'.6]! 9tonomi f"ngsional ini kem"dian

dik"atkan pada pasien dengan progresif eksternal

ophthalmoplegia' menimpan penghap"san skala besar'

termas"k +)' dalam s"b*pop"lasi mereka

molek"l mt#$%! In sit" hibridisasi per0obaan

meng"ngkapkan ak"m"lasi fok"s dihap"s mt#$%

dan transkrip L*strand dengan penipisan bersamaan

transkrip *"ntai di otot : serat dari pasien

[.8'.-]! asil 0on : rm f"ngsional

independensi promotor transkripsi di&i&o!

+eb"ah did"ga sit"s inisiasi ked"a "nt"k *"ntai transkripsi

terletak sekitar 638 n"kleotida posisi

(IT/ pada gen t1$%)he' berbatasan langs"ng

"nt"k gen ,/+ r1$% (4br! /! )romotor na

wilaah hana men"n"kkan kesamaan terbatas dengan ,*bp

Ur"tan konsens"s dan sit"s ini lebih sering dig"nakan

dari IT, "nt"k transkripsi dari *"ntai

[3- ^ .,'.3]!

Meskip"n ada gambaran ang 0"k"p rin0i dari

elemen 0is*a0ting ang terlibat dalam mt#$% transkripsi'

pengetah"an tentang trans*a0ting n"klir dikodekan

faktor masih bel"m lengkap! 5raksinasi biofisik

ekstrak transkripsi mitokondria man"sia memiliki

meng"ngkapkan keb"t"han setidakna d"a trans*a0ting

protein; inti 1$% polimerase relatif non*selektif

dan faktor transkripsi ang tdk

mengan"gerahkan promotor selekti&itas pada polimerase

[.7'im inti diharapkan "nt"k berinteraksi

dengan +) dan L+) sebagai m"tasi pada domain ini

melenapkan bahkan tingkat terendah transkripsi[.6]! #limerase &% mitkndria belum

puri ed untuk mgenitas. &amun demikian" 6D&% manusia

menentukan mitkndria &% plimerase baru-baru ini

mengidenti!ikasi ed le pemutaran urutan diungkapkan

tag (4T) database dengan urutan ragi

[,]! Menarikna' setengah ?*terminal dari prediksi

: signi "r"tan asam amino tidak bisa saham polipeptida

identitas dengan polimerase s"b"nit 1$% t"nggal

T3' T7 dan +)6 bakteriofag!

Man"sia tdk faktor transkripsi akting

dalam konser dengan inti mitokondria 1$% polimerasetelah p"ri : ed [/'3]' ang 0#$% telah


22/40

kloning dan sek"ensing [.] dan gen ang telah

ditandai [ ^ 7]! 5aktor' sekarang diseb"t

mtT5% [8]' adalah berlimpah /*k#a mitokondria

protein dan sebagian besar terdiri dari d"a mobilitas tinggi

kelompok (M4 domain dipisahkan oleh asam amino /7*

linker resid" dan diik"ti oleh resid" asam amino /*dasar ?*terminal ekor! M4 domain dianggap

"nt"k terlibat dalam #$% mengikat dan

ditem"kan dalam seb"ah kel"arga ang agak beragam protein ang

anggota telah terlibat dalam proses seperti

peningkatan transkripsi dan kemasan kromatin

[-]! %nalisis m"tasi dari mtT5% man"sia memiliki

men"n"kkan bahwa ekor ?*terminal penting

"nt"k spesifik : pengak"an 0 #$% dan sangat penting "nt"k

mensponsori tingkat tinggi tertent" : 0 inisiasi transkripsi

[6


23/40


24/40

%T) [77]! The etidi"m bromida bar"*bar" diamati

dan modi %T)*dependent : kation protein*#$%

eak kaki h"l" IT,' ang dapat berkorelasi

dengan per"bahan tingkat r1$% sintesis tetapi

b"kan dari sintesis m1$%' dan indikasi protein"ria seb"ah

+it"s interaksi #$% h"l" IT/ telah; Eerikanen keper0aaan lebih lan"t "nt"k model [6/]! $am"n demikian'

adalah diD0"lt membaangkan bagaimana d"a peristiwa inisiasi mengambil

menempatkan k"rang dari ,


25/40

transkripsi oleh mer"pakan penghalang fisik'

b"kan oleh tertent" : 0 interaksi dengan mitokondria

1$% polimerase' karena mtT=1M "ga

menengahi terminasi transkripsi oleh heterolog

)olimerase 1$% [83]!

@ed"a ?laton dan kelompok %ttardi ini telah ditampilkanbahwa sebagian polipeptida dari lisat mitokondria

mengand"ng polipeptida dengan berat molek"l

sekitar 3. k#a memiliki kemamp"an "nt"k menghasilkan tertent" : 0

#$ase +aa tapak di sit"s pem"t"san terkait

dengan f"ngsi mtT=1M [8/'8]! ?#$% dari

polipeptida dominan dari fraksi ini bar"*bar" ini

kloning dan sek"ensing [86]! )olipeptida

berisi d"a daerah dasar ang terpisah dan

tiga motif >ipper le"sin ang terb"kti

diperl"kan "nt"k : 0 kapasitas tertent" ang #$%*binding [86]!

Aeak ang dihasilkan oleh protein rekombinanadalah ser"pa tapi tak sama dengan ang dihasilkan oleh

5raksi polipeptida 3.*k#a! 1ekombinan protein

"ga terb"kti dapat mempromosikan transkripsi

terminasi dalam sistem in &itro [86]! Ini

)engamatan men"n"kkan bahwa komponen tambahan

dari fraksi polipeptida 3.*k#a diperl"kan "nt"k

akti&itas terminasi! @ompleksitas elas ini

dari mtT=1M tidak mengherankan' mengingat bahwa sehar"sna

dapat memod"lasi akti&itas dalam menanggapi

permintaan sel"lar "nt"k r1$% mitokondria' pada

sat" sisi' dan "nt"k t1$% mitokondria dan

m1$% di sisi lain!

Menarikna' seb"ah heteroplasmi0 % ke 4 transisi di

tengah mtT=1M mengikat sit"s (%3/.34 adalah

sering ditem"kan pada pasien dengan mitokondria

en0ephalomopath M=L%+ (miopati mitokondria'

en0ephalopath' asidosis laktat dan stroke*seperti

episode [87] dan pada pasien dengan maternal diwariskan

diabetes dewasa [88]! #alam st"di &itro memiliki

men"n"kkan bahwa m"tasi ini se0ara dramatis meng"rangi

aDnit dari mtT=1M "nt"k sit"s ang mengikat dan penebab0a0at penghentian transkrip [8/'83'8-]! +ebalikna'

mapan tingkat transkrip h"l" dan

hilir sit"s pem"t"san tidak a2e0ted

oleh transisi %3/.34 di sel k"lt"r [8-'-


26/40

ang hadir dalam &ertebrata mt#$% dan intergeneti0

"r"tan ang minimal' pengolahan polisistronik panang

Ut"san dan L*"ntai did"ga

menadi proses ang relatif sederhana' hana memb"t"hkan beberapa

en>im! 4en t1$% H ank d"a r1$%

gen dan hampir setiap gen protein (4ambar! ,! Iniorganisasi genetik ang "nik telah menebabkan proposal

bahwa str"kt"r sek"nder dari "r"tan t1$%

memberikan tanda ba0a dalam pemba0aan

Informasi mt#$% [/7]! )re0ise eksisi endon"0leolti0

dari t1$% dari transkrip ang bar" lahir akan

bersamaan menghasilkan r1$% diproses dengan benar dan'

dalam banak kas"s' benar diproses m1$% [/7'-3]! #i

kas"s*kas"s di mana m1$% termini tidak bisa

dipertangg"ngawabkan oleh t1$% eksisi (misalna "t"san

"nt"k s"b"nit I sitokrom 0 oksidase [/7'-3]F 4ambar!

,' en>im pengolahan m"ngkin mengak"i sek"nder+tr"kt"r ang berbagi fit"r penting dengan

str"kt"r da"n semanggi khas t1$%!

)ematangan t1$% mitokondria melibatkan

tiga kegiatan en>imatik ang bar"*bar" identi*

: ed oleh 1ossmanith dan rekan [-.] dalam in &itro

eLa sel mitokondria sistem pengolahan t1$%!

)er0obaan mereka men"n"kkan bahwa belahan dada di

)*end mendah"l"i bahwa pada 3)*end! =ndon"klease ang

bertangg"ng awab "nt"k 3)*akhir pembelahan bel"m ditandai!

)embelahan pada )*end dilak"kan oleh

mitokondria 1$ase ) (mt1$ase )! =n>im

mengand"ng pe0ahan ang disiapkan oleh 1ossmanith dan

rekan [-.] memotong prek"rsor t1$% mitokondria

di benar )*end' tapi' tidak seperti persiapan

oleh orang lain (lihat' misalna [-]' tidak membelah t1$%Tr prek"rsor

=s0heri0hia 0oli dengan benar! al ini men"n"kkan

bahwa persiapan sebel"mna terkontaminasi dengan

isoform sitosol dari 1$ase ) ang tampakna

mamp" se0ara ak"rat memproses prek"rsor t1$% bakteri!

1agi mt1$ase ) telah ditandai se0ara rin0i!

=n>im dari +a00harom0es 0ere&isiae terdiridari protein n"klir dikodekan dan mt#$%en0oded

+pesies 1$% [-6'-7]! 1$% bagian dari

kompleks ribon"0leoprotein adalah %U*kaa dan bent"k

inti katalitik dari en>im! )erbandingan

mt1$ase ) 1$% dari spesies ragi di2erent memiliki

meng"ngkapkan &ariasi ang l"ar biasa dalam "k"ran dari .-< ke

,.< n"kleotida [-8]!

)ematangan t1$% ang dipotong selesai oleh

+elain dari "r"tan ??% "nt"k mereka 3)*end katalis

oleh %T) (?T); t1$% n"0leotidltransferase

[-.]! M1$% mitokondria poladenlated olehpoli mitokondria (% polimerase selama ata" segera


27/40

setelah pembelahan [--,


28/40

dapat berspek"lasi bahwa beberapa protein mitoribosomal

telah mengadopsi peran str"kt"ral dan f"ngsional dari

hilang r1$% tetapi data eksperimen "nt"k f"ngsi

dari mitoribosomal mamalia protein tidak

tersedia dan terbar" kema"an pen"l"han

dari sifat*sifat indi&id" mitoribosomalprotein berasal dari st"di di +! 0ere&isiae [,


29/40

d"a terpendek menatakan frame pemba0aan terb"ka dari

mamalia mt#$% (%T)ase8 dan $#.LF 63


30/40

it" bar"*bar" men"n"kkan bahwa @)AM*T"*Ts

kompleks akan memisahkan dengan adana 4T) dan

t1$% berm"atan [,3,]!

.!3! The e2e0t m"tasi titik t1$% pada mt#$%

ekspresi

M"tasi patogen dalam gen mitokondria ang"m"mna heteroplasmi0' ait" tipe liar dan m"tan

mt#$% berdampingan dalam sel ang sama! ild tpe

mt#$% mem"ngkinkan m"tan mt#$% dengan sebalikna

per"bahan dasar mematikan "nt"k bertahan! Maternal mewarisi'

en0ephalomopathies mitokondria sering

terkait dengan m"tasi titik dalam mitokondria

gen t1$% [,3/]! Eiopsi otot dari pasien

men"n"kkan 0a0at histokimia fok"s sitokrom 0

oksidase terkait dengan distrib"si non*a0ak

m"tan dan wild tpe mt#$% [.-'-,]! Mosaik ini

ekspresi penakit ini "ga diamati pada primerk"lt"r sel dari pasien ini seperti digambarkan dalam

4ambar! 3!

@ema"an bar" pada m"tasi mitokondria t1$% bagaimana

mengerahkan e2e0t mereka sebagian besar berasal dari eksperimen

di mana mt#$% pasien telah dipindahkan

"nt"k sel kontrol E

sintesis protein [8-'-


31/40

0brids dibandingkan dengan tingkat dewasa ,6+

r1$%' tingkat men"n"kkan korelasi terbalik ang k"at

dengan tingkat kons"msi oksigen dari 0brids

(indikator fosforilasi oksidatif mitokondria

kapasitas [,38,3-]! )engamatan ini memiliki

menebabkan hipotesis [-


32/40

mengandalkan mikroskopis elektron dan sentrif"gal

analisis mt#$% molek"l dari berb"daa

sel" menun8ukkan baa mlekul mtD&% mamalia

direplikasi unidire6tinall3 dari dua spasial dan

tempral 3ang berbeda" untai-spesi!ik 6 asal *2,. Te

asal replikasi H-strand (imatis diproses"nt"k menghasilkan dewasa primer 1$% 3)*termini!

@arena lokasi mereka' telah berspek"lasi

bahwa ?+E I' II dan III mengarahkan pembelahan tepat

transkrip primer "nt"k memberikan primer ang ses"ai

spesies [36]! )enelitian terbar" di &itro transkripsi

dari wilaah 9 dengan mitokondria 1$% polimerase

5raksi [,.8] men"n"kkan bahwa 1$% prek"rsor

primer ada sebagai 1$%*#$% stabil dan persisten

hibrida "ga dikenal sebagai 1*lingkaran! )embent"kan hibrida

memb"t"hkan "ns"r ?+E II dan "ga a2e0ted oleh

m"tasi pada ?+E III!?ari oleh kelompok ?laton "nt"k akti&itas katalitik


33/40

transkrip mamp" memproses L*"ntai mengand"ng

9 "r"tan menebabkan mengidentifikasikan : kation en>im

diseb"t pengolahan 1$% mitokondria

endon"klease (1$ase M1)F Ulasan di [,.-]! #i

st"di awal mereka' di mana "ntai t"nggal 9 mengand"ng

+pesies 1$% dig"nakan sebagai s"bstrat' dalamsit"s pembelahan &itro 1$ase M1) tidak ses"ai dengan

sem"a in &i&o )*""ng man"sia bar" lahir dan mo"se

*helai (lih [,.6,.7,


34/40

sit"s heterod"pleks' nam"n' tidak menelaraskan

dengan sem"a prediksi di sit"s priming &i&o! )enghap"san

gen homolog dalam +! 0ere&isiae tidak tampak

"nt"k a2e0t mt#$% metabolisme [,6]! #engan demikian' kesimp"lan

"nt"k f"ngsi endon"klease 4 di mitokondria

Eiogenesis m"n0"l premat"r!#alam &ertebrata' peristiwa sintesis ang paling *"ntai kios

tak lama setelah inisiasi! #itangkap bar" lahir *helai

tetap anil ke template mereka L*"ntai dan men0iptakan

str"kt"r tripleks # loop [,.]! The 3)*""ng

premat"r dihentikan *helai n"kleotida peta im mitokondria ang berbeda dari besar

primase #$% n"klir! )rimase #$% mitokondria

dikaitkan dengan 1$%! )engobatan dengan ribon"klease

+eb"ah menebabkan inakti&asi 0epat akti&itas primase dan

men"n"kkan peran penting "nt"k 1$% terkait!

#ominan 1$% bagian 0o*fraksinasi denganakti&itas primase adalah prod"k gen n"klir !8+

r1$% [,73]! )e0ahan $am"n' karena hana mentah

#$% mitokondria primase telah dianalisis'

0o*fraksinasi dari !8+ r1$% m"ngkin telah

hasil dari kontaminasi ad&entif!

!.! 5aktor trans*a0ting terlibat dalam elongasi dan

pematangan helai ket"r"nan

#$% polmerase N adalah sat"*sat"na polimerase #$%

hadir dalam mitokondria [,7.]! 4angg"an dari

4en #$% polimerase N di +! 0ere&isiae telah men"n"kkan

bahwa en>im ang diperl"kan "nt"k sintesis mt#$%

tetapi tidak memiliki f"ngsi dasar l"ar organel

[,7]! #$% polimerase N men"mbang hana ke0il

sebagian ke0il dari total akti&itas polimerase #$% sel"lar'

nam"n sifat en>imatik ang "nik' ketahanan

"nt"k aphidi0olin dan kepekaan terhadap dideoksin"kleosida

trifosfat memb"atna relatif m"dah "nt"k mengidentifikasi

polimerase #$% mitokondria [,76]! =n>im

telah diisolasi dari man"sia [,77] dan beberapa

organisme lain (Ulasan di [,.-]! #$% polimerase

N tampakna rentan terhadap degradasi proteolitikdan str"kt"r s"b"nit ang masih menadi bahan perdebatan!

+ebagian besar data terbar" men"n"kkan bahwa en>im adalah heterodimer seb"ah

pada &ertebrata terdiri dari ,/ ^ ,.


36/40

telah men"n"kkan bahwa s"b"nit "tama adalah e&ol"si

dilestarikan dan telah mem"ngkinkan identifikasi : kation ked"a

polimerase dan eCon"0lease b"kti*memba0a

domain! +t"di kinetik bar"*bar" ini dengan rekombinasi ang

menatakan' ,.


37/40

replikasi!

Tipe I topoisomerase mengkatalisis relaksasi

s"perkoil #$%' sedangkan tipe II topoisomerase adalah

bertangg"ng awab "nt"k pengenalan s"perkoil! Topoisomerase

meng"bah topologi #$% oleh transientl

melanggar obligasi ba0kbone' mengg"nakan mekanismemelibatkan baik "ntai t"nggal (tipe I ata" ganda

strand (tipe II belahan dada [,-3]! Mito0hondriall terkait'

tipe I topoisomerase telah diidentifikasi : ed

dari berbagai s"mber' termas"k le"kemia man"sia

sel [,-.] dan trombosit man"sia [,-]! Mitokondria

en>im im"nologis terkait dengan n"klir

rekan [,-6,-7] dan ragi mito0hondriall terkait

enis kegiatan topoisomerase I dihap"skan

ketika gen ang mengkodekan enis n"klir I topoisomerase

tergangg" [,-8]! Meskip"n elas

im"nologi dan genetik h"b"ngan antaratipe I topoisomerase n"klir dan mitokondria' ang

en>im dapat dibedakan dari masing*masing lain oleh

Tanggapan di2erential "nt"k reagen kimia dan inhibitor'

baik dalam ragi dan man"sia [,-,-7]!

Mito0hondriall terkait tipe II topoisomerase

memiliki telah diidentifikasi : ed di #i0tosteli"m dis0oide"m

[,--]' hati tik"s [/im n"klir! )engobatan sel berb"daa dengan

0ipro H oCa0in menebabkan istirahat "ntai ganda di

mt#$% dan hasil hilangna genom mitokondria

[/


38/40

pengak"an dari "r"tan #$% [/'6'7,] dan

komparatif dalam organello dan footprinting &itro

)enelitian men"n"kkan bahwa mtT5% man"sia mengikat pada terat"r

inter&al spasi sel"r"h wilaah


39/40

faktor trans*a0ting terb"kti lebih diD0"lt karena

konsentrasi sel"ler ang rendah dan potensi risiko

kontaminasi dengan akti&itas en>im analog dari

asal n"klir ata" sitosol dalam per0obaan fraksinasi sel!

$am"n' beberapa mitokondria relatif melimpah

en>im dapat p"ri : ed "nt"k homogenitas dan"r"tan 0oding man"sia mereka telah diklon memanfaatkan

teknik biologi molek"ler kon&ensional!

Ear"*bar" ini' masalah dengan p"ri : kation memiliki

telah dielakkan dengan skrining database =+T man"sia

dengan "r"tan gen ragi ang terlibat dalam transkripsi

dan replikasi [,]! %kibatna' beberapa k"n0i

en>im ang terlibat dalam transkripsi dan replikasi

man"sia mt#$% kini telah dikloning dan dipetakan

ke kromosom (Tabel ,! $am"n demikian' pelengkap

strategi ang diperl"kan "nt"k mengkarakterisasi

repertoar pen"h en>im ang terlibat dalam ekspresi mt#$%sebagai perbedaan e&ol"si antara faktor

dapat men0egah s"kses 0bers0reening dan tertent"

faktor bisa menadi "nik "nt"k mitokondria mamalia

sistem!

)asien dengan kesalahan bawaan replikasi mitokondria'

transkripsi ata" sintesis protein hadiah

menantang serta s"mber daa "nt"k ahli biokimia! The

Tantangan adalah "nt"k memahami mekanisme molek"ler

penakit' sedangkan s"mber daa adalah sebagai alami

teradi m"tan ang memberikan kesempatan "nt"k

0iri aspek proses ini pada man"sia

ang diD0"lt "nt"k mempelaari dengan 0ara lain! )asien

dengan m"tasi mt#$% menediakan tak tertandingi

wawasan ke dalam peran elemen 0is*a0ting! #engan

+eh"b"ngan dengan faktor trans*a0ting ang terlibat dalam pemeliharaan

dan replikasi mt#$%' sat" kelompok pasien

telah menarik perhatian kh"s"s! )ada tah"n ,--,' Moraes

dan rekan [/,6] dielaskan beberapa bai

dengan penipisan ditandai mt#$% dan oksidatif 0a0at

fosforilasi! +eak penem"anna' lebih

dari 3< pasien telah dielaskan dengan sindrom ini(dibahas dalam [/,7]! @ebanakan pasien datang

segera setelah lahir dengan kelemahan otot dan hati

kegagalan ata" t"b"lopath ginal terkait dengan berat

menipisna mt#$% (88 ^ --J dalam aringan a2e0ted di

post*mortem! Tidak ada m"tasi mt#$% telah

didok"mentasikan pada bai ini dan analisis silsilah

konsisten dengan warisan a"tosomal dari sifat terseb"t!

+eb"ah keterlibatan genom n"klir telah : 0on rmed

di d"a kel"arga dengan teknik transfer mt#$%

[/,8,33]! ilangna progresif mt#$% pada aringan

"ga diamati pada k"lt"r sel dari beberapa pasien[/,8,33]! #alam b"daa menipis' sebagian ke0il


40/40

sel masih mengand"ng kadar tampakna normal

mt#$% tapi mt#$% replikasi k"rang lebih berk"rang

di sem"a sel (4ambar! .! +e0ara bersama*sama' ini

pengamatan sangat menarankan penangkapan replikasi karena

ke : efisiensi de dari trans*akting' faktor n"klir dikodekan!

)asien dengan tampilan penipisan mt#$% men"r"ntingkat mtT5% dalam aringan [/im k"n0i dari replikasi mt#$% m"ngkin akan

mematikan di awal perkembangan embrio [/inkan pengembangan alat diagnostikserta wawasan patogen bar" "nt"k meminimalkan

konsek"ensi ang menghan0"rkan dari ekspresi 0a0at

genom mitokondria!

4oogle Translate for E"siness;Translator Toolkitebsite Translator4lobal Market 5inder

T"rn off instant translation%bo"t 4oogle TranslateMobile?omm"nit)ri&a0 O

Termselp+end feedba0k
http://www.google.com/url?rs=rsmf&q=http://translate.google.com/toolkit%3Fhl%3Denhttp://www.google.com/url?rs=rsmf&q=http://translate.google.com/manager/website/%3Fhl%3Denhttp://www.google.com/url?rs=rsmf&q=http://translate.google.com/globalmarketfinder/%3Flocale%3Denhttps://translate.google.com/?hl=en&eotf=0http://www.google.com/url?rs=rssf&q=http://translate.google.com/about/intl/en_ALL/http://www.google.com/url?rs=rssf&q=http://www.google.com/mobile/translate/http://www.google.com/url?rs=rssf&q=//translate.google.com/communityhttp://www.google.com/url?rs=rssf&q=http://www.google.com/intl/en/policies/http://www.google.com/url?rs=rssf&q=http://www.google.com/intl/en/policies/http://www.google.com/url?rs=rssf&q=http://www.google.com/support/translate/http://www.google.com/url?rs=rsmf&q=http://translate.google.com/toolkit%3Fhl%3Denhttp://www.google.com/url?rs=rsmf&q=http://translate.google.com/manager/website/%3Fhl%3Denhttp://www.google.com/url?rs=rsmf&q=http://translate.google.com/globalmarketfinder/%3Flocale%3Denhttps://translate.google.com/?hl=en&eotf=0http://www.google.com/url?rs=rssf&q=http://translate.google.com/about/intl/en_ALL/http://www.google.com/url?rs=rssf&q=http://www.google.com/mobile/translate/http://www.google.com/url?rs=rssf&q=//translate.google.com/communityhttp://www.google.com/url?rs=rssf&q=http://www.google.com/intl/en/policies/http://www.google.com/url?rs=rssf&q=http://www.google.com/intl/en/policies/http://www.google.com/url?rs=rssf&q=http://www.google.com/support/translate/

biomol transe

Documents