functional importance of mobile ribosomal proteins

7/24/2019 Functional Importance of Mobile Ribosomal Proteins

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assembl% ?11G10@ but also un(ins mR&3 structures uring translation ?1@6 4scherichia coli mutantslac8ing other r-proteins, incluing !1, !11, !00, ., an .> proteins, remain *iable ?10, 1, 1@6'herefore, these r-proteins ha*e been chosen as fluorescence labeling sites in single-molecule FHrsterresonance energ% transfer 9smFR4': e;periments that unra*el the translational %namics of theribosome ?1, 1E@6 'he !1# protein is a special case (here it is the onl% r-protein to e;ist as multiplecopies of imers on the ribosome ?1>@, but onl% one imer is re=uire for the cell to sur*i*e, albeit at a

lo(er gro(th rate ?#+@6 'he !> protein is e*en more intriguing (here espite its conser*ationthroughout bacteria, the !>-eletion 46 coli mutant oes not sho( substantial efects in cell gro(th ?#1,##@6 !> is absent in archaeal an eu8ar%otic ribosomes ?#0@, inicating that it is actuall% ispensable formost of the translational acti*ities6 In fact, ## out of r-proteins are sho(n to be nonessential (henelete ini*iuall% in 46 coli ?#@6 'his raises se*eral curious =uestions incluing (h% these proteinsare preser*e uring the course of e*olution an (hether their absence irectl% impacts theconformational %namics of the ribosome or the% associate (ith cellular functions through moreinirect (a%s6

.tructures of most of the r-proteins ha*e been (ell resol*e in +. ribosome comple;es, (ith somee;ceptions incluing the .1 protein an the !1 an !1# stal8s (hich are highl% mobile an oftenmissing in -ra%-sol*e structures6 Despite the gro(ing repertoire of ribosome structures fromifferent species (ith no*el techni=ues, structures of the r-proteins !1# an .1 ha*e ne*er been full%etermine in comple; (ith the ribosome6 Interestingl%, .1 not onl% has high conformational fle;ibilit%?#, #@ but also associates (ea8l% (ith the ribosome ?#@6 'he unstructure &-terminal omain 9&'D:of .1 fols upon bining to the ribosome in a (a% similar to man% intrinsicall% isorere proteins9IDPs: ?#E, #>@6 .ince there has been an increasing interest in the foling an functionalit% of IDPs, ansince the ribosome also e;ploits the conformational fle;ibilit% of some r-proteins for factor recruitmentan moulation of protein s%nthesis, here (e re*ie( some of the bacterial r-proteins, namel%, .1, !>,!1 stal8, an !1# stal8 9Figure 1:, (hich lac8 structural information an ma% function through theirintrinsic fle;ibilit%6Figure 15 Ribosomal components iscusse in this paper6 Moeling of the ribosome is escribe inMethos6 protein6 reen5 !1 stal8, compose of the!1 protein an /GE 9nucleotie #+>0G#1>: of #0. rR&36 Magenta5 !1# stal8, incluingnucleotie 1+0+G11# of #0. rR&3 an r-proteins of !1+, !11, an !1#6 Details of !1# stal8 areepicte in Figure 6 Due to the fle;ibilit% of .1, onl% a fragment of the .1 &'D in comple; (ith .# isresol*e b% -ra% 9PD$ '@:, an the rest of .1 is represente b% o*al6 /ere, the .1 protein isnot inclue in the ribosome moel, an the interaction bet(een .1 &-terminal heli; an .# isinicate b% ashe line6#6 .1 Protein Does &ot 3l(a%s .ta% on the Ribosome but Participates in arious Functions


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phage-encoe N-subunit, .1 is one of the four subunits of the ON phage R&3 replicase holoenB%me?0>, +@6 It also associates (ith the N protein from phage to form a component of generalrecombination ?1@6 It promotes enB%matic acti*ities incluing transcriptional c%cling in *itro ?#@ anR&3-clea*age b% the ' phage enoribonuclease Reg$ ?0@6 'he *ersatilit% of .1 seems to be enableb% multiple 0G#1>: part of the stal8 is sho(n anhighlighte6 3n open conformation 9green, PD$ #I#' ?0+@: is foun in the ribosome lac8ing an 4-sitetR&3 9re:6 When the tR&3s are in their 3QP an PQ4 h%bri states, !1 stal8 aopts a full% closeconformation 9magenta, PD$ 0RE. ?01@:6 In the presence of a classical-state 4-site tR&3, !1 stal8resies in bet(een those conformations an thus becomes half-closeS 9blue, PD$ 0IEI ?0#@:6 'he 3-site 9%ello(:, P-site 9orange:, an 4-site 9re: tR&3s in their classical states an the #0. rR&3 9gra%:are ta8en from PD$ 0IEI ?0#@6

3fter peptie bon formation, the 0+. rotates G1#T countercloc8(ise relati*e to the +. (hen *ie(efrom the sol*ent sie of 0+. 9terme rotate stateS: ?, @6 Ribosome translocation is then facilitateb% fluctuation of tR&3s bet(een the classical 3Q3 an PQP states an the h%bri 3QP an PQ4 states 9theletter before slash enotes the site (here the anticoon-stem loop 93.!: bins in 0+., (hile the otherletter is the site (here the acceptor stem bins in +.: ?E@6 'he mobilit% of the !1 stal8 ma% irect themo*ement of a eac%late tR&3 from PQ4 state to 4Q4 state ?>, +@, (here the tR&3 issociatesspontaneousl% ?1@6 Pre*ious structural stuies sho(e that (hen the 4-site is *acant 9nonrotateribosome:, the !1 stal8 aopts an open conformation 9e6g6, PD$ #I#' ?0+@:6 When a PQ4-state tR&3 ispresent 9pretranslocational rotate ribosome:, the stal8 is full% close to interact (ith the tR&3 9e6g6,PD$ 0RE. ?01@:6 3 half-close stal8 is obser*e (hen an 4Q4-state tR&3 is present in aposttranslocational nonrotate ribosome 9e6g6, PD$ 0IEI ?0#@: 9Figure #:6 .ingle-molecule FR4'e;periments further re*eale that the !1 stal8 fluctuates bet(een open an close configurations in thepretranslocational ribosome, an that !1 stal8 opening is strongl% suppresse after bining of 4F-?#@6

'he !1 protein has se*eral basic resiues to form salt briges (ith the aciic tR&3 bac8bone in PQ4state, but it forms less salt briges (ith the initiator than (ith the elongator 6 'he !1 stal8 rR&3 alsohas a (ea8er stac8ing interaction (ith PQ4-state ?>@6 'a8en together, the !1 stal8 has a lo(er affinit%for as compare to , an it opens more fre=uentl% in the presence of a PQ4-state ?#@6 Interestingl%,(hen all moifications of the ribonucleosies in PQ4-state 9Figure 09a:: are e;clue uring molecular%namics 9MD: simulations, Domain II of !1 protein seems to become *er% fle;ible an mo*einepenentl% of Domain I ?>@6 'herefore, both the ientit% an the chemical constituents of PQ4-state


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tR&3 affect the mo*ement of !1 stal86 'he lo(er affinit% (ith !1 stal8 an the resulting slo(ertranslocation 8inetics of initiator 9(ith properl% moifie ribonucleosies: ma% help to stabiliBe theinitiation comple;6 Moifie ribonucleosies are also important for the functions of elongator tR&3s,such as 9Figure 09b::6 'he% stabiliBe tR&3 foling ?0@ an moulate tR&3 bining (ith !1 stal8 ?>@6Figure 05 &atural R&3 moifications ?00@ in 99a: PD$ #FM' ?0@: an 99b: PD$ 0IUW ?0@:6 ms#i3V #-meth%lthio--isopenten%laenosine2 m V -meth%lguanosine2 D V ih%rouriine2 V

pseuouriine2 m) V -meth%luriine2 s) V -thiouriine2 Cm V #-@6 It remainsa m%ster% (h% the ribosome re=uires multiple copies of !1# imers 9e=ui*alent to P1QP# imers ineu8ar%otes ?@: to achie*e optimal initiation an elongation efficienc% ?#+@6

'he !1# C'D is responsible for interacting (ith translation factors ?@6 .ince !1# C'Ds are highl%mobile an e;ist as multiple copies of imers, !1# C'Ds ha*e been propose to recruit an eli*erelongation factors 4F-'u an 4F- to the ribosomal factor bining site b% increasing the encounterfre=uenc%, an thereb% leaing to association rates higher than e;pecte for ranom collisions ?@6

3fter initial encounter (ith a translation factor, such as IF#, 4F-'u, 4F-, or RF0, the !1# C'D ma%facilitate loaing of the translation factor into the factor bining site Aointl% (ith !11s &'D through aconser*e proline s(itchS mechanism ?E@6 It has been emonstrate that 4F- can ri*e cis-transisomeriBation of the proline s(itch 9P.##: on !11 through the pepti%l-prol%l cis-trans isomerase9PPIase: center locate bet(een the -omain an Domain of 4F-6 'he cis form of P.## enables


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the !11 &'D to interact an immobiliBe the !1# C'D, an thereb% allo(ing full accommoation ansubse=uent 'P h%rol%sis of 4F-6 P.## is then s(itche offS to the trans form possibl% b% DP-boun 4F- through an un8no(n mechanism ?E@6 'hus, the 4F-, functioning as a 'Pase, PPIase,an a translocase that promotes translocation of the translation comple;, facilitates its o(n bining tothe ribosome b% inirectl% altering the mobilit% of the !1# C'D6 In contrast, (hen the intrinsicmobilit% of !1# C'D is restricte b% shortening the fle;ible loop bet(een &'D an C'D, the

translation acti*it% is comparable to that of !1#-eplete ribosomes, but oubling the length of lin8erhas limite effects ?>, +@6 'he proline s(itch mechanism ma% be uni*ersall% conser*e for othertranslational 'Pases in all three omains of life ?E@66 $acterial !> Protein Is Conser*e an %et &onessential for 'ranslation

'he bilobe architecture of !> protein consists of a globular &'D oc8ing into #0. rR&3, a long heli;lin8er, an a globular C'D ?1@ 9Figure 1:6 In all cr%stal structures of the (il-t%pe ribosome, !>e;tens its C'D far a(a% from the ribosomal surface an contacts (ith the 0+. subunit of aneighboring ribosome6 Depletion of !> leas to ifferent cr%stal forms, (hich allo(s resol*ingribosomes in comple; (ith translational 'Pases ?#@6 Inee, !> aopts a istinct bent conformationto(ar the . protein in a cr%o-electron microscop% 9cr%o-4M: structure ?0@6 &otabl%, althoughelongation factors are occlue b% the neighboring ribosomes !> in cr%stal pac8ing, both open 9seen in-ra% structures: an bent 9seen in the recent cr%o-4M structure: conformations of !> o not actuall%clash (ith nearb% elongation factors in the organiBation of pol%somes ?0, @6 Whether !> coorinatespol%some formation b% briging neighboring ribosomes remains un8no(n6

'he functional role of !> in reaing frame maintenance is most iscernible uring e;pression of 'phage gene+6 'he gene contains a b%pass region (here the ribosome recogniBes the nascent peptiesignal an the mR&3 hairpin an then hopsS a +-nucleotie gap before resuming translation ?, @6'he hop-1 mutation, (hich is a .er>0Phe alteration in the !> C'D, is foun to partiall% restoreb%passing efficienc% in the absence of a stable gene+ hairpin6 Interestingl%, hop-1 mutation oes notincrease bac8(ar frameshifting efficienc%, but complete epletion of !> increases both for(arslippage an bac8(ar slippage6 'herefore, !> is propose to bloc8 bac8(ar slippage b% posing asteric hinrance bet(een neighboring ribosomes, (hile for(ar slippage ma% be suppresse b% specificinteractions bet(een the !> C'D an the upstream neighboring ribosome ?@6

In aition to the phage-specific gene, massi*e occurrence of programme translational b%passingelements 9b%ps: is foun in mitochonria ?E@6 'hese b%ps ma% originate from intron-li8e mobilegenetic elements ?>@6 .ubse=uentl%, phages ma% contribute to e*olutionar% i*ersification of bacteriab% propagating these mobile b%ps6 Consiering the e;tensi*e coe*olution of bacteria an phages ?+@,an the possible bacterial origin of mitochonria, their ribosomes ma% e*ol*e to ensure propertranslation of genes bearing b%ps6 Interestingl%, no !> homolog has been foun in eu8ar%otic E+.ribosome ?#0, 1@6 'his ma% be ue to the facts that !> has more prominent functions uringtranslation of phage-specific b%ps an that eu8ar%otic cells are much less epenent on *irus fori*ersification of gene pools6

.imilar to .1, !> ma% participate in cellular processes other than translation6 3lthough !> eletionmutants o not e;hibit appreciable gro(th phenot%pes, mutations in the essential ribosome biogenesis'Pase Der protein cause epenence on !>6 For the 'hrIle mutation, (hich impairs the 'Paseacti*it% of Der, !> epletion leas to an aberrant, elongate cell morpholog% an a efect in celli*ision6 Interestingl%, !> oes not rescue the 'Pase acti*it% of Der in *itro, suggesting that !> ma%not irectl% interact (ith Der to complement the efecti*e phenot%pes6 .ince !> &'D, (hich bins to#0. rR&3, is sufficient to complement the er mutant, !> ma% share a similar function (ith Der in


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promoting anQor stabiliBing correct assembl% of the +. ribosome ?#1@6 /o(e*er, the preciseph%siological functions of !> an Der remain to be unco*ere66 3 Mechanical ie( of the Ribosome an r-Proteins

Intrinsic %namics of a protein are encoe in the topolog% of its nati*e contacts ?#G@6 4lasticnet(or8 moel 94&M:, a coarse-graine *ersion ?G>@ of normal moe anal%sis ?E+, E1@ 9Figure :,

has been e;tensi*el% use since the mi->+s to stu% the intrinsic %namics of biomolecules ?, E#,E0@, especiall% for supramolecular protein 9or proteinQnucleic aci: assemblies ?EGEE@6 In 4&M, themolecular structure of interest is coarse-graine to the resiue le*el as noes, (ith interactions bet(eenthese noes being appro;imate b% a simple harmonic potential ?G>@6 'a8ing anisotropic net(or8moel 93&M:, the most broal% use 4&M, as e;ample, the potential ta8es the form/ere, is the uniform spring constant an is the number of noes in the net(or82 an are the istancesbet(een the th noe an the th noe at an instantaneous moment an at the e=uilibrium state 9obtainefrom e;perimentall% sol*e structures:, respecti*el%6 'he /ea*isie step function, , e=uals 1 for noepairs (ith separation shorter than a cut-off istance 9i6e6, : an e=uals Bero other(ise6 Preicte thermalmotions, in the form of a fluctuation matri; 9or interchangeabl% referre as co*ariance matri;S:comprising noe-noe 9auto:correlations, can be eri*e from(here is a -imensional isplacement *ector an for noes in 0-imensional space6 is the $oltBmannconstant, an is the absolute temperature6 is the /essian, a force constant matri; encoe b% proteincontact topolog% at e=uilibrium ?, E#, E0, E>@6 'he co*ariance matri;, eri*e from the in*erse of/essian, can be further ecompose into the sum of an orthonormal basis set, the normal moes6 'heresulting an from eigen*alue ecomposition are the th smallest eigen*alue an the corresponingeigen*ector, respecti*el%6 'he first si; eigen*alues are e=ual to Bero, corresponing to egrees offreeom for rigi-bo% rotation an translation in 0-imensional space6 4ach is a form of *ibrationalmotion that the biomolecule can perform 9the th normal moe:, (ith its fre=uenc% being the s=uare rootof 6Figure 5 Coarse-graining of emeth%lase 3l8$ 9PD$ &I/: in 4&M6 For each amino aci, the CLatom is ta8en as the representati*e noe6 'hree noes, namel%, the P atom of the phosphate group, theC# atom of the nitrogenous base, an the C atom of the pentose, are chosen to represent a nucleotie6'he ifference in the number of noes reflects the fact that the a*erage molecular (eight of each aminoaci is X11+ Da, (hile that of each nucleotie is X00+ Da6 /ere the simple harmonic potentials bet(eennoes (ithin a cut-off istance of 1 Y are enote b% lines6Figure 5 '6 thermophilus #0. rR&3 color coe accoring to $-factors from + Y# to + Y# ?0#@6 For*isual contrast an clarit%, $-factors larger than + Y# are all colore re6 &ucleoties of P'C,incluing 3#1, )#+, )#E, C##, an 3#+#, are sho(n as green spheres6

4&M has been applie to refine structures ?>+, >1@ an e;tract resiue-le*el information from electronparamagnetic resonance 94PR: ?>#@, smFR4' ?>0@, an cr%o-4M ?>@6 It is also inispensable to stu%supramolecules %namics such as the ribosome6 Molecular %namics 9MD: simulations ?>@, apo(erful chemical techni=ue that (as e*elope since the +s an enAo%e a ela%eac8no(legement (ith &obel PriBe a(are in #+10, ha*e pro*ie escriptions of ribosomal%namics up to a couple of hunre nanosecons ?>, >@6 'he time scale is ho(e*er a fe( orer ofmagnitues shorter than, sa%, the (ell-8no(n ratcheting motion of the ribosome that is characteriBee;perimentall% b% -ra% ?>E@ an cr%o-4M ?@ an 8no(n to occur on the timescale of millisecons tosecons6 >@6It therefore confirms that ratcheting motion, a relati*e rotation bet(een 0+. an +. subunits, isintrinsic at room temperature an encoe mainl% b% rR&3s contact topolog% ?#@6 Furthermore,resiues at the mR&3 entrance of the ribosome (hich e;hibit correlate motions (ith the mR&3 (erereail% re*eale b% 4&M an (ere propose to be the acti*e sites of the ribosomal helicase ?#@, some


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of (hich ha*e alrea% been supporte e;perimentall% ?1@6 It is note(orth% that protein %namicspreicte b% 4&M generall% locate 8no(n catal%tic resiues an oc8ing interfaces aroun*ibrationall% an rotationall% inert regions, ma8ing efficient preictions of functional sites possible ?,E>, 1++@6 'his tenenc% can be e;plaine b% the fact that a preorganiBe an rigi catal%tic site isre=uire to pro*ie stabiliBing en*ironment for the transition state of the substrate ?1+1G1+0@6

i*en that high-resolution cr%stal structures of the ribosome are alrea% a*ailable in great etail, themagnitue of thermal motion for each resiue can be straightfor(arl% obtaine from e;perimental $-factors6 3s e;pecte, the pepti%l transferase center 9P'C: is burie in a rigi region of #0. rR&3 (ithlo( $-factors 9Figure :6 $efore peptie bon formation, the ribosome is loc8eS in the nonrotatestate to promote catal%sis ?1+, 1+@6 3lso, P'C ma% be locate aroun the rotational a;is of the +.subunit, so that the thermall% ri*en rotational motion bet(een 0+. an +. subunits is furtherminimiBe6 .ince the rigi-bo% rotational a;is of an obAect is etermine b% its center of mass 9CM:,(e ne;t as8 (hether P'C lies in pro;imit% to the CM of +. subunit 9:6 3s escribe in Methos, here(e moel all missing resiues an subunits in the '6 thermophilus +. ribosome 9PD$ F: ?0#@ b%homolog% moeling 9Figure 1:6 3s sho(n in Figure , the P'C, (hich consists of 3#1, )#+,)#E, C##, an 3#+# of #0. rR&3 ?1+, 1+@, is in pro;imit% to the calculate 9re sphere:6Consiering the fact that man% r-proteins ecorate the rR&3 core on its peripher% (ithout essentialfunctions, it coul be that the r-proteins ma% fine-tune the mass istribution of the ribosome in orer toachie*e optimal tR&3 translocation an peptie bon formation, as suggeste b% Wang an Jerniganpre*iousl% ?1+E@6 /ere (e further elaborate this iea b% consiering the mass balance bet(een !> an!1# proteins that lie on the opposite sies of the +. subunit6

First, (hen the outermost !1# imer is remo*e along (ith its bining segment of !1+ C'D ?1>@, tiltsto(ars !> 9Figure , blue sphere:6 is elete, lies closer to the !1# stal89Figure , green sphere:6 While multiple !1# imers are re=uire for efficient factor recruitment, !>ma% be important for counterbalancing the mass contribute b% multiple !1# imers6 In the absence of!>, rotation of the subunits, tR&3 translocation, an peptie bon formation ma% be slightl%compromise6 'his ma% be the case uring translation of the b%p in gene+, (here !> eletionincreases the propensit% of ribosome slippage6 Conse=uentl%, (e e;pect that eleting one cop% of !1#imer shoul lea to similar phenot%pe of reuce frame maintenance, (hich coul be partiall% rescueb% remo*al of !> 9Figure , gra% sphere:6 Despite the conser*e o*erall architecture of ribosomes, therR&3 cores from '6 thermophilus an 46 coli iffer slightl%, (hich ma% lea to ifference in the cop%numbers of !1# imers bet(een the t(o species6 3n interesting possibilit% is that e*en thoughincreasing the length of !1+ C'D, an conse=uentl% the number of accommoate !1# imers, ma% bea*antageous for recruitment an acti*ation of elongation factors ?@, the loss of mass balance ma%compromise translation fielit% anQor spee6Figure 5 Deletions of r-proteins affect 6 Calculation of is e;plaine in Methos6 Re sphere5 (il-t%peposition of 6 $lue sphere5 mutants , (here the outermost !1# imer an its boun !1+ C'D segmentare elete 9right inset:6 reen sphere5 of the !> eletion mutant6 ra% sphere5 of the compensator%ouble mutant (ith aforementione eletions6 &ucleoties of P'C 93#1, )#+, )#E, C##, an3#+# of #0. rR&3: are sho(n as stic8s6

3lthough !> an !1# are =uite istant from P'C that irect interactions seem unli8el%, consiering theregulator% roles of !1# imers in the proper functioning of 4F-'u an 4F-, the possibilit% remainsthat the% act inirectl% through interactions (ith elongation factors6 i*en that the ribosome cans%nthesiBe oligopepties (ithout elongations factors an 'P, albeit at a *er% slo( pace ?1+>, 11+@,one ma% use such factor-free in *itro s%stem to probe ho( the presence an absence of !> as (ell asifferent cop% numbers of !1# imers regulate translation (ithout interferences from other factors6 'o


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further eluciate ho( the ifferences in mass istribution ma% alter local %namics aroun P'C, onecan appl% 4&M to stu% the 3-site an P-site tR&3s %namics for *arious ribosome mutants lac8ing orgaining subunits of !> an !1# imers6 'he preictions can then be compare (ith smFR4'e;periments, (here 3-site an P-site tR&3s are fluorescentl% labele ?111@6 'he h%pothesis that r-proteins 9especiall% !> an !1# stal8: ma% act b% balancing the mass of the ribosome therefore callsfor e;perimental *aliations6 'he mass-balancing arrangements of subunits ma% be a general scheme

for regulating catal%tic efficienc% in enB%mes, (hich is a esirable feature for rational esign of usefulenB%mes66 Methos616 Moeling the Missing .ubunits an Resiues of the Ribosome

'he elongation comple; from 'hermus thermophilus 9PD$ F: ser*e as the starting template forthe +. ribosome moel6 Missing subunits an resiues (ere moele b% superimposing homologousstructures from the PD$ atabase, follo(e b% a #+-step energ% minimiBation (ith R

functional importance of mobile ribosomal proteins

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