Origin and Evolution of the Ribosome - CSHL...

Origin and Evolution of the Ribosome

George E. Fox

Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001

Correspondence: [email protected]

The modern ribosome was largely formed at the time of the last common ancestor, LUCA.Hence its earliest origins likely lie in the RNA world. Central to its development wereRNAs that spawned the modern tRNAs and a symmetrical region deep within the large ribo-somal RNA, (rRNA), where the peptidyl transferase reaction occurs. To understand pre-LUCAdevelopments, it is argued that events that are coupled in time are especially useful if one caninfer a likely order in which they occurred. Using such timing events, the relative age ofvarious proteins and individual regions within the large rRNA are inferred. An examinationof the properties of modern ribosomes strongly suggests that the initial peptides made by theprimitive ribosomes were likely enriched for L-amino acids, but did not completely excludeD-amino acids. This has implications for the nature of peptides made by the first ribosomes.From the perspective of ribosome origins, the immediate question regarding coding iswhen did it arise rather than how did the assignments evolve. The modern ribosome isvery dynamic with tRNAs moving in and out and the mRNA moving relative to the ribosome.These movements may have become possible as a result of the addition of a template to holdthe tRNAs. That template would subsequently become the mRNA, thereby allowing the evo-lution of the code and making an RNA genome useful. Finally, a highly speculative timelineof major events in ribosome history is presented and possible future directions discussed.

A major commonality of all cellular life isthe coupling between translation and tran-scription mediated by the genetic code. Com-parative genomics has further refined this byrevealing the presence of an “RNA metabolism”(Anantharaman et al. 2002) or “Persistent pro-teome” (Danchin et al. 2007) that is basicallya compendium of essentially universal genesinvolved in translation, transcription, RNAprocessing and degradation, intermediary andRNA metabolism, and compartmentalization.DNA replication likely arose later because the

core enzymes involved in the process are notrelated (Bailey et al. 2006 and others). Togetherthese universal genes comprise what is fre-quently referred to as LUCA, the last universalcommon ancestor (Benner et al. 1993; Lazcano1994; Mushegian and Koonin 1996; Kyrpideset al. 1999). It is noteworthy that no matterhow they are defined, by far the largest numbersof genes in LUCA are associated with trans-lation. Indeed, the translation machinery asrepresented in LUCA is essentially completeindicating that major events in its origins

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occurred before LUCA. Thus, it might appearthat the origins of the translation machinerywould be hopelessly obscured by time. Never-theless, as will be discussed herein, substantialalthough necessarily incomplete, evidence re-lating to the origins and early development ofthe translation machinery and its relation toother core cellular processes continues to existin the primary sequences, three-dimensionalfolding, and functional interactions of the vari-ous macromolecules involved in the modernversions of the translation machinery.

The modern ribosome consists of small andlarge subunits (30S and 50S in Bacteria andArchaea) that come together during the initia-tion of protein synthesis remain together asindividual amino acids are added to a growingpeptide according to information encoded onthe mRNA, and finally separate again in con-junction with the release of the finished protein.Each subunit is an RNA/protein complex. InBacteria and Archaea, the 50S subunit typicallycontains a 23S rRNA and a 5S rRNA whereasthe 30S subunit contains the 16S rRNA. Peptidebond synthesis occurs in the 50S subunit at thepeptidyl transferase center, (PTC), and codonrecognition occurs at the decoding site, whichis in the small subunit. Transfer RNAs, (tRNA),bridge the two subunits occupying, at varioustimes in the synthesis cycle, the A, P, or E(exit) sites of the 50S subunit and the decodingsite in the 30S subunit. A universal CCA se-quence at the 30 end of the tRNA is the pointof attachment of the amino acid and later thegrowing peptide chain to the tRNA. The A, P,and E sites are partly in the small subunit andpartly in the large subunit such that a tRNAcan be in a hybrid site (e.g., the A site in the30S and P site in the 50S. The mRNA is exclu-sively found in the small subunit where it inter-acts with the anticodon loops of the tRNAs. Asthe nascent protein is synthesized it passesthrough an exit tunnel that begins at the PTCcenter and ultimately exits from the back ofthe 50S subunit. Synthesis is a dynamic cyclicprocess in which tRNAs enter the ribosomebringing amino acids as specified by themRNA and move through the machinery, whichundergoes a series of coordinated motions that

drive the process (Steitz 2008). These includethe movements of the tRNAs between sites,opening and closing of the L1 stalk on the 50Ssubunit and the ratcheting of the small subunitrelative to the large subunit (Frank and Agrawal,2000), which has recently been elucidated instructural detail (Zhang et al. 2009).

Diverse species (Escherichia coli, Haloarculamarismortui, Thermus thermophiles, and Deino-coccus radiodurans) are represented among thevarious atomic resolution ribosome structuresnow available (Ban et al. 2000; Yusupov et al.2000; Wimberly et al. 2000; Schuwirth et al.2005; Selmer et al. 2006; and others). Thesestructures encompass 30S and 50S subunits aswell as the whole 70S ribosome. In addition,cryoelectron microscopy studies have revealeddynamic motions associated with the ribosome(Frank and Agrawal 2000; Connell et al. 2007;and others). These ongoing high resolutionstructural studies provide the opportunity toexamine the relative age of features within theribosome such as the A, P, and E sites, the exittunnel, the L7/L12 region, and the L1 regionthat facilitate the entry and exit of tRNAs.

PEPTIDYL TRANSFERASE CENTER

It is believed that the peptidyl transferase center,(PTC), which encompasses the large subunitportions of the A and P sites of the ribosome, isstructurally the same in both the 50S and 70Ssubunits (Steitz 2008). When comparing 50Ssubunit structuresbetween ArchaeaandBacteriaone again finds that the structures are essentiallythe same. However, the E site structure is differ-ent. In Archaea L44e interacts with the E-sitetRNA but this protein is missing in Bacteriawith the result that the tRNA CCA end is posi-tioned differently. Hence, the A and P sites likelypredate the E site, which may have been addedpost-LUCA (Steitz 2008).

The portion of 23S rRNA comprising thePTC contains a region of approximately 165bases that shows high twofold pseudo symme-try (Agmon et al. 2005; Zimmerman & Yonath2009). The two 82 nucleotide halves of the sym-metrical region correspond to the 50S portionof the A and P sites of the ribosome. In fact,

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the essence of this region is contained in a singlecontiguous self-folding RNA (Smith et al. 2008).The PTC is located in Domain 5 of the 23SrRNA structure.

Recently, Hsiao et al. (2009) superimposedthe structure of the large subunit RNAs fromtwo ribosome crystal structures and sectionedthe resulting structure into concentric shellswith the PTC at the center. They, like others(Ban et al. 2000; Wimberly et al. 2000), foundthat ribosomal proteins (r-proteins) are effec-tively absent from the PTC region, which iswhy the ribosome is regarded as fundamentallyan RNA machine. To the extent that proteinelements are in proximity to the PTC, they areshort, largely unstructured peptides ratherthan globular elements. The globular regionsare mainly on the surface of the ribosome(Ban et al. 2000; Wimberly et al. 2000). A majorstabilizing element in the PTC region is insteadMg2þ interactions. In many cases, the phos-phate oxygen atoms act as inner sphere Mg2þ

ligands (Hsiao et al. 2009; Hsiao and Williams2009). Thus, consistent with the notion of a pre-ceding RNA world, the structure of the PTCseems to have evolved before the availability ofproteins.

Although the modern translation machin-ery is very complex, two small RNAs, the PTCRNA fragment and tRNAs are at its core. Bothof these are less than 100 nucleotides in length,and their importance supports the notion thatthe translation machinery was originally a dis-covery of the RNA world. In fact, the ability tosynthesize coded peptides of increasing com-plexity would eventually terminate the RNAworld and create the RNA/protein world. Theseldom discussed issue is whether such a termi-nation would have occurred before (e.g., briefRNA world) or after the discovery of an RNAreplicase (extended RNAworld). If peptide syn-thesis arises quickly, then their will neither betime nor need for extensive catalysis of bio-chemical reactions by RNA. If reasonable, therapid appearance of a translation system mayeven eliminate the need to validate the RNAworld by demonstrating the self-replicatingRNA system that has proven experimentallydifficult to achieve.

tRNA ORIGINS AND INCREASING RNACOMPLEXITY

Because of its obvious importance, considerableattention has been focused on the origins of thetRNA and numerous models have been pro-posed and recently reviewed (Di Giulio 2009).The most popular model (Noller 1993; Maizelsand Weiner 1993 and 1994; Schimmel et al.1993; Schimmel and Henderson 1994), envi-sions the tRNA as having two domains, eachencompassing half the molecule. One domaincontains the terminal CCA sequence to whichthe incoming amino acid or growing peptideis attached. The second domain contains theanticodon and associated loop that interactwith the mRNA. The two domains are fre-quently envisioned as being of different agewith the CCA domain being older. Supportfor this idea stems from the fact that the CCAdomain alone forms a “minihelix” to whichmodern tRNA synthetases can readily attachspecific amino acids. Such aminoacylation hasalso been shown with evolved ribozymes (Leeet al. 2000), which can be surprisingly small(Chumachenko et al. 2009). In fact, aminoacy-lation has been reported without any enzymeor ribozyme at all (Tamura and Schimmel2004). Furthermore, it has also been reportedthat a minihelix when incorporated into the50S subunit can participate in peptide bondformation (Sardesai et al. 1999). Indeed, eventhe addition of a single cytosine (equivalent toC75 of modern tRNAs) to puromycin is appa-rently sufficient to allow peptide bond for-mation (Brunelle et al. 2006). Thus, it mayinitially only be necessary to have the CCA seg-ment alone (Nissen et al. 2000). The 50 domainof the tRNA is not consequential to peptidebond formation and could have been addedlater. If the tRNAs evolved from the one domainstructure or an even simpler structure, then pro-tein synthesis would likely have begun as anoncoded process (Schimmel and Henderson1994). Single domain or even smaller aminoa-cylated RNAs are especially attractive in anRNA world where synthesis of larger RNAs islikely to be difficult. Synthesis of randomoligomers in the 20–40 size range has been

Ribosome Evolution

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shown (Joshi et al. 2009; Powner et al. 2009;Szostak, 2009; Ferris et al. 1996) but the pathto prebiotic synthesis of large RNAs is not with-out difficulties (Orgel, 2004).

How does one obtain RNAs of increasingcomplexity, such as those of modern tRNAs orthe PTC RNA, without a true RNA replicase?There are two core possibilities, ligation andhybridization. RNA ligation has been shownto be feasible in an RNA World (Hager et al.1996; Hager and Szostak 1997; McGinness andJoyce 2002). Thus, it is of interest that the tRNA“cloverleaf” secondary structure can be formedby a direct duplication, e.g., ligation, of an ap-propriate stem loop structure (Di Guilio2002). The possible relevance of this idea wasenhanced further by the demonstration that itwas possible to actually replicate all the major ter-tiary interactions seen in modern tRNAs whentwo appropriate stem loop structures were ligatedtogether (Nagaswamy and Fox 2003).

An alternative method of readily obtainingmore complex structures is to simply hybridizesmall fragments to one another such that alarger RNA with many “nicks” is assembled.These nicks might or might not be sealed at alater stage. In Nanoarchaeum equitans, severaltRNAs are encoded as partially complementaryhalf molecules, which are then ligated togetherto form a tRNA (Randau et al. 2005a and b).In Euglena gracillis the large subunit rRNA iscomprised of 14 discrete RNA fragments heldtogether by hybridization events that formvarious helical elements. Not only are the frag-ments not coded in the order they appear inthe final RNA but they are actually intermingledin the genome with similar fragments of thesmall subunit RNA (Smallman et al. 1996).

Chirality and the Ribosome

In modern organisms, mechanisms for non-ribosomal peptide synthesis exist for specializedpurposes and can produce peptides with unus-ual structures and mixed chirality (Marahieland Essen 2009). Modern rRNAs and tRNAsare chiral with D sugars and during translationthey work together to make chiral proteinswith exclusively L-amino acids. This is highly

advantageous to modern organisms becausemixed chirality is clearly undesirable for thesynthesis of structural elements such as a-helices and b-sheets that characterize modernproteins (Bada 2001; Sandars 2005). It is gener-ally assumed that the modern chiral preferencesreflect chiral synthesis of D-ribose in the RNAworld (Tamura and Schimmel 2006; Tamura2008). It thus is likely widely believed thatcharging of the tRNA by aminoacyl tRNA syn-thetases and peptide bond formation from theirbeginnings were chiral. In fact, with atomic res-olution structural data now available, a theoret-ical analysis of the PTC indicates that the naturalchirality of the sugar ring in the RNA iswell paired with the choice of L-amino acids(Thirumoorthy and Nandi 2008).

Nevertheless, it has been shown that D-amino acids can bind to both the A and P sitesof the ribosome in competition with their L-isomers (Quiggle et al. 1981; Bhuta et al. 1981).When elongation tRNAs carrying D-aminoacids are presented to the ribosome in vitrothey are incorporated extremely poorly, butincorporated nevertheless (Yamane et al. 1981;Heckler et al. 1988). It has recently been foundthat peptide synthesis can be effectively initiatedwith D-amino acids (Goto et al. 2008). In addi-tion, by introducing mutations in the PTCregion and/or other nearby regions of the 23SrRNA, it was possible to obtain enhanced toler-ance of D-amino acids in vitro (Starck et al.2003; Tan et al. 2004; Dedkova et al. 2003 and2006). These results suggest that even thoughthe rRNA is itself chiral, the essentially exclusivechirality of the modern ribosome is likely theresult of selection rather than being a funda-mental property of the PTC.

Regardless, of the preference of the ribosomein the modern machinery, D-amino acids willtypically not reach the modern ribosome be-cause the charging reaction also shows a strongbut again imperfect chiral preference. For exam-ple, tyrosyl-tRNA synthetase is able to transferboth D and L tyrosine to its cognate tRNAalthough the L form is significantly preferred(Sheoran et al. 2009). Consistent with the notionthat charging with D-amino acids can occur invivo, the modern cellularmachinery has avariety

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of mechanisms in place to prevent it. Theseinclude deacylases that remove D-amino acidsfrom incorrectly charged tRNAs before they reachthe ribosome (Soutourina et al. 2000; Yang et al.2003). In addition, many aminoacyl tRNA syn-thetases have an editing domain. Nevertheless,the charging reaction has a strong chiral prefer-ence even in minihelix reactions (Tamura andSchimmel 2006; Tamura 2008).

Given that the first tRNA charging processwas unlikely to better than the modern version,the onset of the ribosome as the machine formaking chiral proteins likely emerged not fromthe chiral exclusivity of its processes, but ratherfrom the fact it is a two tiered process with thesame preference at both steps. Thus, if at earliertimes 80% of the tRNAs were charged with an L-amino acid and 80% of the tRNAs charged witha D-amino acid were subsequently excluded bythe ribosome then 96% of the residues incorpo-rated into the growing protein would be of the Ltype. Thus, a modest peptide of 50 amino acidswould perhaps have only two D-amino acids andthus have a good chance of being functional. Infact, incorporation of a D-amino acid into amodern protein is not necessarily destructiveto the protein, but instead depends on where itoccurs (Dedkova et al. 2003, 2006).

In summary, given that the modern ribo-some is not exclusively chiral the early peptidesynthesis machinery likely had significantlyless chiral specificity. This would be true evenif its RNA components were exclusively chiralas the various editing mechanisms associatedwith the modern charging process would nothave been available. Thus, the peptides madeby early ribosomes likely included D-aminoacids and hence would tend to be unstructured.

Would such peptides be useful? Probablyyes. For example, small, largely unstructuredpeptide segments are found in what are likelythe older (not oldest) areas of the modern ribo-some. Once the two tier chiral selection pro-cedure used in the modern ribosome wasestablished, refinements in either aspect couldquickly improve the likely use of the productpeptide. Future studies of partially chiral pep-tides might provide better insight to the natureof the earliest peptides and clarify the extent of

chiral preference in the ribosomal machinerythat is needed to begin to produce reasonablenumbers of peptides of the modern type.

RNA HISTORY—INFERRING EVENTSBEFORE LUCA

The PTC and tRNAs clearly existed beforeLUCA. The fact that we can infer likely if notproven aspects of their history suggests that wecan learn about events that occurred beforeLUCA. But, can we do this in a more generalway? A key step is recognition that there aremany opportunities to gain insight into relativetiming. For example, if a ribosomal protein,(r-protein), is modified after translation by anenzyme, then there is a timing associationbetween the two proteins. The more difficultsecond step is to develop evidence pertainingto the relative age of the associated entities,e.g., which is older? In the example, one mightinitially speculate that the modifying enzymeis newer than its target protein because withouta target what good would the modifying enzymebe? However, this might not be the case becausethe modifying enzyme may have been recruitedfrom somewhere else after the emergence of ther-protein. Finally, the two proteins could haveemerged essentially simultaneously.

Given such essentially opposite alternatives,how can they be resolved? The answer willtypically be independent information or a likelyassumption. If the r-protein is found in all organ-isms and the modifying enzyme is only in Gram-positive bacteria, then it is more likely, but notproven, that the r-protein is older. Not provenbecause the gene for the modifying protein mighthave been lost in other lineages. If the protein inquestion is associated with a duplication event, asis the case with ribosomal proteins L15 and L18e,it may be possible to use phylogenetic argumentsto deduce relative age (Roberts et al. 2008). In theexample used here, when one examines the struc-ture of the two proteins, the r-protein is muchsmaller and far simpler, composed only ofa heli-ces. Thus, the growing evidence for one hypoth-esis makes it increasing likely to be correct.Finally, the third step is to find more extensiveassociations involving multiple timing events.

Ribosome Evolution




An examination of the evidence relatingto elongation factor G (EF-G) illustrates howtiming information might be combined into amore general hypothesis. It has been arguedfrom atomic resolution structures that EF-Gis a structural mimic of elongation factor EF-Tu when it is part of a ternary complex withGTP and an aminoacylated tRNA (Nissen et al,1995; Moore, 1996). If true, this hypothesis raisesthe obvious question of who mimics whom?Because the tRNA is at the core of the translationmachinery, it likely arose very early. From this,one can logically argue that EF-G is likely newer.Regardless of the status of molecular mimicry,EF-G is actually not completely essential forthe translocation process (Gavrilova & Spirin,1971; Gavrilova et al. 1976; Spirin, 2002). Is thetiming relationship proven? No, but at this stageit appears more likely than the alternative. Otherclaims of mimicry in the ribosomal machineryhave also been made (Selmer et al. 1999; Naka-mura and Ito, 2003).

The nonobvious benefit of the mimicryargument is that one can combine it with thepreviously discussed argument that tRNAs be-gan as one domain RNAs (Schimmel and Hen-derson, 1994; Di Guilio, 1994; Schimmel andRibas de Pouplana 1995). Thus, one producesa combined core time-line for the developmentof the translation machinery from a one domaintRNA predecessor, to a two domain tRNA, fol-lowed by the addition of EF-Tu, and finally theaddition of EF-G. However, EF-Tu has beenshown in at least one case to bind to a modelone domain (minihelix) tRNA (Rudinger et al.1994) which supports a hypothetical time linethat delays the onset of the second tRNA untilafter the emergence of EF-Tu. This is interest-ing, because it also delays the possibility ofmRNA-tRNA interaction and thus the onsetof coding. However, in the absence of codingit is not clear how one produces a sophisticatedprotein.

DEDUCING THE RELATIVE AGE OFr-PROTEINS

Given that the ribosome is quite ancient, onemight have expected the early r-proteins to have

diverged to spawn later ones and possibly evensuper families of proteins used elsewhere(Ohnishi 1984; Leijonmarck et al. 1987). Infact, there are clear examples of genetic eventssuch as gene fusion, insertion and duplication(Ramakrishnan and White 1998), but in generalmost r-proteins are structurally distinct. Somelikely evolved with the early ribosome and thenin some cases were recruited to other functions,whereas others likely evolved elsewhere and gotincorporated into the ribosome at later stages.Thus, there are likely to be historical relation-ships between some of the proteins that will pro-vide timing insights to the development of thesubunits. One such example, e.g., S6 and S10,has been uncovered (Jue et al. 1980) and verifiedby structural data (Brodersen et al. 2002).

Can one deduce the relative age of thevarious r-proteins? Phylogenetic distribution isan obvious initial indicator with the morewidely distributed proteins likely being older.However, many r-proteins are universal in allthree Domains of life (Lecompte et al. 2002;Hartman et al. 2006), and hence it is not imme-diately obvious how one might infer relative ageamong members of this rather large group.Also, one must be alert to the fact that like theRNAs, all parts of the r-proteins are probablynot equally old (Vishwanath et al. 2004). Exper-imental studies have shown that ribosomalcomponents are assembled in a reproduciblemanner, which might recapitulate to a signifi-cant extent the history of the ribosome andthereby provide timing information. For thisreason, it was hypothesized that the oldest pro-teins would assemble first and be at the core ofthe process whereas newer proteins would beincorporated into later stages of assembly andthe newest proteins would be last (Fox and Naik2004). The process of in vivo assembly is cur-rently being actively studied at a very detailedlevel (Klein et al. 2004, Nierhaus 2007). How-ever, traditional maps (Rohland and Nierhaus1982; Herold and Nierhaus 1987; Nierhaus1991) that summarize in vitro assembly remaina reasonable approximation of what actually oc-curs. Consistent with the hypothesis regardingassembly, an initial inspection of the traditionalmaps shows that the nonuniversal and hence

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likely newer r-proteins are largely incorporatedinto the ribosome at the final stages of assembly.

To focus on what are likely the oldest pro-teins, all the nonuniversal proteins and theirassociated connections were removed fromthe 50S subunit assembly map (Fig. 1). Thehypothesis here is that the assembly order ofthe remaining universal r-proteins speaks totheir relative age. Thus, although L23 bindsdirectly to 23S rRNA, its assembly is also facili-tated by L3 and hence it is likely a neweraddition than L3. Another aspect of Figure 1is that essentially all the remaining r-proteinsare still interconnected but some are more con-nected than others. In complex systems, greaterinterdependence is likely to be associated withlonger association and hence suggests greaterage. For example, L1 and L3 are both universaland directly interact with the RNA, however,L3 is more central to the process of assemblyand hence likely older.

Genomic organization can also be consid-ered. Universally conserved gene clusters (e.g.,operons) are very rare. When genes are associ-ated in conserved operons they are likely toshare regulatory relationships. In the case of r-proteins, four clusters of r-proteins (the S10,Str, Spc and L13 operons) are preserved in theArchaea and Bacteria (Siefert et al. 1997). In gen-eral, the universal r-proteins are encoded in theuniversal operons. Thus, r-proteins L2, L3, andL4 are all encoded by the S10 operon. Whenthe assembly information is considered in com-bination with the other criteria, the results sug-gest that L2, L3, and L4, are among the oldestr-proteins (Fox and Naik 2004; Tran et al. in prep-aration). Overall, the conserved large subunitproteins have initially been grouped into fourclusters ranging from oldest to most recent.These four groups are (1): L2, L3, L4; (2): L22,L23, L24; (3): L5, L6, L10, L11, L13, L18, L29;and (4): L9, L31, L32-L34.

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Figure 1. Assembly map of 50S ribosomal subunit with all nonuniversal protein omitted. The map was derivedfrom Nierhaus (2001) and is a slightly modified version of that presented previously Fox and Naik (2004). Eachprotein is indicated by a numbered box with the 23S rRNA indicated at the top. Lines with arrows indicate orderin assembly with darker lines representing stronger dependencies. Thus L4 and L24 bind directly to the RNA andwork together to facilitate the incorporation of L22. Boxes are colored with regard to the similarity of theirposition in assembly. For example, yellow indicates terminal proteins, which are not required for addition ofany other universal protein.

Ribosome Evolution




INFERENCES FROM r-PROTEINSTRUCTURE

Detailed examination of the structure of theolder r-proteins and how they interact withthe rRNAs is likely to provide insight to thedevelopment of the ribosome before LUCA.There are large amounts of information in thisregard and to illustrate what might be learnedtwo interesting examples, S1 and L2 will be dis-cussed in some detail. Ribosomal protein L2 isuniversally distributed, plays a central role inribosome assembly, is encoded in the universalL10 operon, and is near the PTC although notinvolved in peptide bond synthesis. Analysisof the assembly map discussed earlier suggestsL2 is in fact one of the very oldest proteins. L2has a RNA binding domain comprised of anOB-fold and an SH3 -like barrel.

The SH3 domain is homologous to similardomains found in the NusG protein (involvedin Rho dependent termination of transcrip-tion), and two r-proteins, L24 (universal) andL21e (not universal). Although L24 is universalit is actually only required to initiate subunitassembly (Spillmann and Nierhaus 1978), Inactuality this role can be assumed by L20 atlow temperatures (Franceschi and Nierhaus1988) and a mutant E. coli strain defective inL24 is viable (Herold et al. 1986). L21e andL24 are thus likely newer than L2 even thoughL24 binds directly to the rRNA.

The OB-fold is found in r-proteins S1, S12,S17, and S28e. The OB-fold is a small b-barrelformed from 5 strands connected by modulat-ing loops; two or three loops on the same faceof barrel are consistently observed acting asclamps to bind to their ligands (Agrawal andKishan 2003). The SH3 domain has a character-istic fold with b-barrel architecture, whichconsists of five or six b-strands arranged astwo tightly packed antiparallel b sheets. Twoprominent loops, termed the RT and n-Srcloops, are often seen in the fold (Boggon andEck 2004). What is especially interesting fromthe perspective of ribosome origins is that thesetwo folds are actually very similar. In particular,the insertion of strand b1 between b4 and b5in the SH3-fold would actually create an

OB-fold like topology (Agrawal and Kishan2001). Thus, not only is L2 a possible progenitorof multiple r-proteins, its modern version mayhave arisen as a result of a very early (preLUCA) duplication event creating two copiesof one of the folds followed by a rearrangementin one of the domains to create one fold of eachtype. If this is correct, the obvious next questionis which folding domain is older? In fact, theSH3 domain is encompassed entirely within auniversal sequence block (Vishwanath et al.2004) whereas the OB fold is partially in a blockthat distinguishes Bacteria and Archaea. Thisobservation suggests that the SH3 domainmay be older although position in the ribosomeshould also be considered.

Ribosomal protein S1 is substantially largerthan all other r-proteins and in contrast withL2 is not integrally part of the ribosome. It isinvolved in initiation and has been associatedwith antitermination and trans-translation aswell. It lacks an Archaeal homolog and is some-times missing even in Bacteria suggesting it ispost-LUCA addition to the ribosomal machi-nery. S1 contains six copies of an RNA bindingdomain (OB fold) that is known as the S1domain. Many proteins in fact have one ormore S1 domains. These include, but are notlimited to: Polynucleotide phosphorylase, abacterial exonuclease that degrades mRNAfrom 30 to 50 (Regnier et al. 1987); the a subunitof the eukaryotic initiation factor 2 (Gribskov,1992); yeast PRP22, an RNA helicase likeprotein required for the release of the mRNAfrom the spliceosome (Company et al. 1991);and the amino-terminal end of ribonucleaseE, which is involved in both 5S rRNA processingand the rapid degradation of mRNA in E. coli(Kaberdin et al. 1998). Perhaps, the most nota-ble of the proteins that contain S1 domains forthe present purposes are the translation initia-tion factor IF1 and its eukaryotic equivalenteIF1a, both of which also have the characteristicfive stranded b barrel arrangement (Sette et al.1997; Battiste et al. 2000). The proteins contain-ing S1 domains can be broadly grouped intothree main functional groups of RNA process-ing, involvement in transcription or translationand chromatin or septum regulation. The S1

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motif is found in all three domains of life withthe IF-1/eIF1A type are universally distributedsuggesting this might be the original source ofthe fold. It seems likely that ribosomal proteinS1 is a late addition to the ribosome, possiblyderived from the initiation machinery.

EVOLUTION OF THE LARGERIBOSOMAL RNA

The substantial structural and sequence conser-vation seen in comparisons of the rRNAs fromall three Domains of life suggest that theyreached their modern size early in the develop-ment of the ribosome. However, it is not neces-sarily true and in fact it is extremely unlikelythat all parts of the rRNAs are of the same age.Instead, like some of the r-proteins, they haveincreased in size over time, perhaps beginningas an amalgamation of smaller fragments (Clark1987; Gray and Schnare 1996). Indeed, theeukaryotic RNAs are tolerant of insertions incertain locations and have clearly grown largersince LUCA (Gray and Schnare 1996; Yokoyamaand Suzuki 2008). Thus, cogent arguments havebeen presented that certain portions of therRNAs are older than others (Gray and Schnare1996; Wuyts et al. 2001; Mears et al. 2002;Caetano-Anolles 2002; Hury et al. 2006).

The secondary structure of the large rRNAreveals the presence of six domains in theRNA with the PTC being located in DomainV. Hury et al. (2006) argue that interconnectiv-ity among distant regions can provide insight tohistorical timing. The argument is that olderregions would have more time to be integratedinto the structure and hence would show greaterconnectivity to other regions than newer addi-tions. To implement this timing argument, allof the base–base interactions between regionsthat were not contiguous in the secondary se-quence were counted. A number of regionswere identified as being the most connectedand hence likely to be the oldest (Fig. 2).

The oldest regions largely overlap with theminimal RNA previously deduced by compara-tive analysis (Mears et al. 2002) and theobserved minimal core (Gutell 1992; Gray andSchnare 1996). In fact, rRNAs comprised of

essentially only the regions highlighted in Fig-ure 2 are found in various minimize mitochon-drial rRNAs such as the large subunit RNA ofTrypanosoma brucei (Sloof et al. 1985). Theonly exception is the GTPase region (aboveRegion 2.5 on Fig. 2), which is involved in con-formational changes during protein synthesisand hence not involved in interactions. It wasargued from these results that in addition toDomain V, Domain IV, and a portion ofDomain II (2.1 and 2.3 on Fig. 2) were alsoextremely old but no definitive decision wasmade regarding relative age. Domain IV is ofspecial interest because it has major contactswith the 30S subunit (Yusupov et al. 2001)The addition of Domain IV to the struc-ture likely corresponds with the beginning ofthe formation of the 30S subunit. Hsiao andWilliams (2009) have observed that there arefour magnesium microclusters that are sharedin ribosome structures by the Bacteria (Ther-mus thermophilus) and the Archaea (Haloarculamarismortui). These complex clusters occurfour times in the large subunit. One cluster isexclusively in the PTC whereas two others con-nect parts of Domain 2 (Regions 2.1 and 2.3) toDomain 4 and the PTC.

Recently, Bokov and Steinberg (2009) haveimproved on the connectivity argument byrecognizing the potential of using the many A-minor motifs in the large subunit rRNA as tim-ing events. The A-minor motif occurs when astack of adenosines pack into the minor grooveof a duplex region that can be some distanceaway in the primary sequence of the RNA(Nissen et al. 2001). Such a two-componentinteraction is inherently a timing event, if onecomponent of the interaction is likely to predatethe other. Bokov and Steinberg observed that inmost A-minor interactions involving the PTCregion (very old) the A stack was in the PTCand the helix was elsewhere (presumablynewer). This implied that the A stack usuallypredated the helix it interacted with. From thisassumption, they were able to deduce an orderof addition of individual RNA regions as theRNA grew over evolutionary time, and createda hierarchical map of the RNA. The only otherregion where the A stack portion of the

Ribosome Evolution




interaction is is concentrated is in Domain II(regions 2.1 and 2.3), which suggests that thisregion is also very old and in fact therefore likelyolder than Domain IV. Thus, the PTC region isenvisioned as beginning its expansion beforethe small subunit RNA evolved. Because thedecoding site is in the small subunit, this sug-gests that significant enhancement of the ma-chinery occurred well before the beginning ofcoded peptide synthesis.

The immediate importance of the Bokovand Steinberg (2009) analysis is the ability toderive a hierarchical model of 23S rRNA. Thecore areas of the structure are again the PTCby assumption, the same portions of DomainII (region 2.1 in red on Fig. 2) and parts ofDomain 4 as seen by Hury et al. (2006). How-ever, in the context of studies of ribosome evo-lution, a hierarchical organization is useful in

that it offers the potential for organizing diversedata into a single framework. Thus, one may beable to map the emergence of specific regions ofvarious r-proteins to the emergence of par-ticular rRNA segments and using other datapossibly time adjust the events on differentbranches of the rRNA evolution map. Using thisperspective, Bokov and Steinberg argue that theacquisition of the GTPase center and L1 protu-berance and even the addition of the 30S subu-nit are relatively late additions to the ribosome.These suggestions are largely consistent with thetimeline proposed earlier (Fox and Naik 2004).

RECENT RNA AND PROTEIN COEVOLUTION

An alternative approach to gain insight to howthe RNA can change over time is to studychanges that have occurred since LUCA. The

Figure 2. The secondary structure of Haloarcula marismortui 23S rRNA is broken into six major domains (Ithrough VI) with subregions in the various domains denoted as 1.1, 1.2, etc. The highlighted regions are themost interconnected as measured by the numbers of base-base interactions between a residue in one domainand a residue in another. The figure is taken from Hury et al. 2006.

G.E. Fox




availability of crystal structures of 50S riboso-mal subunits from both Archaeal and Bacterialspecies has provided detailed information aboutthe r-proteins that are unique to either theArchaea or Bacteria and how they interactwith the RNA (Ban et al. 2000; Yusupov et al.2001; Schuwirth et al. 2005). Frequently, aprotein missing in one system is replaced by adifferent protein in the other with the implica-tion that these diversifications developed afterthe divergence of the Bacteria and Achaea(Klein et al. 2004). By examining the relation-ship between these variable r-proteins and theirassociated RNA regions in detail, it is possible todocument the nature of the coevolution that hasoccurred between the r-proteins and rRNA

since LUCA. One might then, with some risk,infer that similar principles applied in thepre-LUCA era.

A detailed examination revealed that thereare few completely unique proteins in eitherDomain of life. Instead, there are many exam-ples of analogs. These have been analyzed indetail from a structural perspective (Kleinet al. 2004). Table 1 summarizes the data foreach of the nonuniversal proteins (Wang,2006). In addition to three Archaeal proteinsthat have no homolog or analog in the Bacteria,there are three examples in which a single Arch-aeal protein has a clear analog in the Bacteriaand five examples in which the Bacterial analogis comprised of two proteins. In each case, a

Table 1. Nonuniversal r-proteins in Archaea and Bacteria.

Arch. Bact. Conserved rRNA interaction region Difference in 23S rRNA

L18e X 23S rRNA domain II H28, H30; domainIII H38, H42

Extra H30 in Archaea

L19e X 23S domain I H34; domain II H47, H53,H57, H58, H60; domain IV H62, H63;domain VI H96

H57, H63 are different for Bacteria andArchaea

L37ae X 23S domain II H28, domain III H56,H58, domain IV H62, H67

H56 is different for Bacteria and Archaea

L39e X 23S domain I H6, H10, domain III H49,H50, H51

No obvious difference

X L25 5S rRNA No obvious differenceX L36 23S domain H42, domain V H89,

domainVI H91, H97No obvious difference

L21e L27 23S domain II H34, domain V H81,H86; 5S rRNA

Slight difference at the end of H86

L24e L19 23S domain VI H96, H101 No differences for RNA, but different L3in Archaea and Bacteria next to L24eor L19

L31e L17 H47, H61, H96, H100 Same secondary and tertiary structuresfor both Archaea & BacteriaL32 C-end H100

L37e L34 23S domain I H5, H8, H23; domain IIH32, H33; domain III H49

No differences for RNA; has interactionwith proteins.

L15e L9 Several residues connecting 23S domainIV H75, H76

Extra H15 in some prokaryotic species,different H10, H79 nearby betweenArchaea and BacteriaL31 23S domain I H11, H13, H15, H21;

domain III H52; domain V H75L32e L20 H2, H25, H40, H41, H45, H46 Different H25 between Archaea and

BacteriaL21 H26, H40L44e L33 H86, H88 H68 (only interacts with L44e) and H88

differ slightly between Arch and Bact.L35 H13, H86, H88

The symbol X indicates no protein is present. Helix numbers are from Yusupov et al. 2001. L7ae is not considered because

although it is not universal it is found in some Gram positive bacteria.

Ribosome Evolution




rather major change in the protein make up inthe ribosome is associated with a very modestchange in the RNA.

As an example, the region of the 23S rRNAthat interacts with L17 and L19 in the E. coli 50Sparticle interacts instead with L24e and L31ein the H. marismortui 50S subunit. Primarysequence and structural comparisons of theseproteins make it clear they are completely unre-lated. Thus, the L17/L24e and L19/L24e pairsare clearly analogs created by convergent evolu-tion. In contrast, the RNA structure is largelythe same in this region in both the Archaeaand Bacteria. Because the RNA structure islargely unchanged, it is likely that the proteincomponents represent independent enhance-ments (probably mainly stabilization) of aneven older RNA. The proteins, however, havelikely been added in the Archaeal and Bacteriallineages since the common ancestor. In somecases, the extra protein is found in only one lin-eage and remains as a “hole” in the other, thusleaving us to speculate whether it has been lostor the use of a having protein at that locationhas only so far discovered in one lineage. In gen-eral, the RNA shows either no structural changeor minimal change, whereas the proteins aredramatically different. In essence, we learn noth-ing about how the RNA grew, but it is very clearthat the proteins at least in the post-LUCA erawere not the driving force.

TIMELINE OF RIBOSOMAL EVOLUTION

To better organize the information regarding theorigins and subsequent history of the ribosomediscussed in the previous sections, it is perhapsuseful to attempt to construct a time-line tooutline a possible sequence of major events inthe context of key historical events (Gray andSchnare 1996; Fox and Naik 2004; Wolf andKoonin 2007). Initial ribosomal developmentwas likely fairly serial, but as its complexityincreased it is probable that many developmentsbegan to occur in parallel, thus making a lineartime line increasingly unrealistic as oneapproaches the post-LUCA age. In what follows,a scenario that attempts to incorporate the var-ious insights discussed earlier is outlined.

The ribosome as envisioned here wouldhave its earliest beginnings in an RNA world.Amino acids or similar molecules would beattached to very small RNA oligomers. Whenthese RNAs encountered one another in thepresence of a RNA ancestral to the PTC RNA,amide bond formation would occur with theresult that larger peptide-like molecules wouldbe created. Such a reaction has been shown tobe in the realm of possibility in an RNA world(Zhang and Cech 1997). These earliest RNAswould be stabilized by Mg2þ. The peptideswould be of mixed chirality but enriched for L-amino acids perhaps as a result of an excess ofD-ribose in the RNAs of the RNA world. Theearly peptides might stabilize various RNAs inthe RNA world and hence be advantageous. Ascomplexity increased single domain tRNAsand the PTC region would emerge. The PTCregion already encompassing the beginnings ofthe exit tunnel would grow, adding first thecore region of Domain II and shortly thereafterportions of Domain IV. At some stage, thedecoding domain of the tRNA will be addedcreating the modern two domain tRNA.

Although there is currently no evidenceaddressing this, the second domain of the tRNAmay have offered the opportunity of anchoringthe tRNA to an accessory RNA thereby increas-ing the amount of time the tRNA is associatedwith the PTC and hence perhaps increasingthe probability of reaction (Wolf and Koonin2007). The introduction of an anchoring RNAwould have been a huge advance. By movingthe anchoring RNA, one could move the prim-itive tRNAs and hence improve their orienta-tions relative the PTC. The growing smallribosomal subunit likely soon took on the taskof moving the template leading to the abilityto eject used tRNAs and encourage arrival ofnew ones. Once such an anchoring RNA exists,the unexpected occurs. The anchoring RNA canserve as a template and later as a true mRNA,making it feasible to develop coded synthesis.

Alexander Mankin (see reviewer commentsto Wolf and Koonin 2007) and perhaps othershave raised the possibility that portions of thesmall ribosomal subunit RNA originated notin later times as an addition to the growing

G.E. Fox




ribosome, but rather separately in the RNAworld where it may have originally served as areplicase. Thus, when recruited to the emergingprotein synthesis machinery, this RNAwould becapable of traversing a template. Many find thismodel attractive as it preserves the notion ofemergence of an RNA RNA replicase in theRNA world.

The key question regarding the genetic codeis not the nature of the assignments, but ratherwhen did a proto mRNA get added to the sys-tem? Decoding is inherent to the small subunitas are many of the movements associated withprotein synthesis. In particular, the ratchetingmotions of the small subunit are largely respon-sible for the movements of the tRNAs amongthe A, P, and E sites. Thus, we need to knowthe order of development of various regions inthe small subunit and how their emergencetracks the development of the large subunit.At present this information is not readilyavailable.

Once a true mRNA and core small subunitmovements are in place, the ribosome wouldbecome increasingly complex by adding earlyconserved proteins such as L2, L3 and L4. Fur-ther expansion of the rRNA could occur by sub-sequent additions, for example the 5S rRNAand its associated proteins. With the onset ofcoding, it would be useful to store information,so an early RNA genome perhaps consisting ofmultiple 10KB or less RNA fragments wouldlikely exist. What would that first genomeencode? Clearly, one possibility is the conservedr-protein clusters, all of which are regulated atthe RNA level (Siefert et al. 1997; Olsen andWoese 1997).

The next major step would be the addition ofthe modern versions of the GTPase center to thelarge ribosomal subunit with a resulting majorincrease in synthesis rates. This would allowa great radiation of cell types and likely end theage of progenotes (Woese and Fox 1977) whilebringing on the post-LUCA age. Consistentwith this late addition of the GTPase centeris the recent argument (Frank and Gonzalez2010) that the ribosome is essentially a Brownianmotor and that EF-G is ancillary rather thaninstrumental in promoting movements.

Further refinements would be ongoing atthis stage such as improvements in initiation,the addition of the exit site, the addition ofL1, which facilitates entrance of tRNAs, intro-duction of posttranscriptionally modified nu-cleotides, and the enzymes that create them etc.The Archaeal and Bacterial RNAs would belargely fixed but newer nonuniversal proteinswould be added and integration between pro-tein synthesis and transcription increased. Ulti-mately limitations on genome size and stabilitywould lead to early RNA genomes being re-placed by DNA genomes.

CHALLENGES AND FUTURE DIRECTIONS

It is clear from what is presented here that muchcan already be inferred about the history of theribosome in times that preceded LUCA. In theearliest stages of ribosome evolution, the cellu-lar entities carrying “protoribosomes” wouldhave lacked a genetic code and the complex dy-namic systems of the modern ribosome. Suchan entity would thus be in the “throes of evolv-ing the genotype-phenotype relationship” andwould be properly considered to be a progenote(Woese and Fox 1977). By the time of LUCA,the ribosome clearly exists in essentially itsmodern form. This strongly suggests that theribosome reached a critical stage of develop-ment that facilitated the final transition fromthe RNA world to the RNA /protein world.What was the causative event in ribosome his-tory? It might be argued that it was coding,but if this were the case the LUCA ribosomewould likely be much more primitive. It shouldinstead be a development that is taking place asthe LUCA ribosome emerges. It is argued hereand elsewhere (Hury et al. 2006; Grela et al.2008) that this key event was the addition ofthe GTPase center to the ribosome. Althoughnot essential to synthesis, the GTPase centerdramatically increases the rate of peptide syn-thesis (Gavrilova and Spirin 1971; Gavrilovaet al. 1976; Spirin 2002). Such an increase mayhave facilitated the transition from an RNAworld to a RNA/protein world.

Looking toward future studies, the evolu-tion of the small ribosomal subunit and its

Ribosome Evolution




RNA are starkly missing from what is presentedhere. There is an assembly map of the 30S sub-unit (Nomura et al. 1984) and efforts to refine itare being actively pursued (Sykes and William-son 2009 and others). It is clear that the headregion, which includes the decoding site isactively involved in the ratcheting motions(Frank and Agrawal 2000) and hence the univer-sal proteins in the 30 domain of the 16S rRNAsuch as S7 are likely among the oldest. Clearlya major next step will be to examine the smallsubunit in detail with particular emphasis onthe dynamic motions that occur during trans-lation. A key to understanding small subunithistory will be detailed knowledge of how andespecially where these structural rearrange-ments occur. Such knowledge is just now reach-ing the literature (Bashan and Yonath 2008;Munro et al. 2009; Zhang et al. 2009) and hasnot yet been digested by the origins community.The small subunit is not the only missing piece.There are other aspects of the story that havenot been addressed here. These include the evo-lutionary development of the aminoacyl tRNAsynthetases, the initiation and terminationaspects of translation, and the maturation andmodification process that the RNAs and to alesser extent the proteins undergo.

There are already substantial amounts ofinformation in the literature regarding theseand other issues which need to be brought to-gether in the near future, perhaps as a commun-ity Wiki site on ribosome evolution similar towhat is being performed for RNA families(Daub et al. 2008). This is especially true foraspects of translation that evolved entirely orin part after LUCA. For example, initiation dif-fers significantly between Bacteria and theArchaea/Eucaryota, but nevertheless severalkey components are shared (Hernandez,2008). Thus, IF-1 and eIF-2 in share an RNA-binding motif with r-protein S1 (Gribskov1992). An examination of the Archaeal uniquer-proteins (Wang et al. 2009) showed thatmany are genomically clustered with genesinvolved in transcription and initiation. In con-trast, the older universal r-proteins are exclu-sively associated with one another with thesingle exception of integration with the core

subunits of the RNA polymerase. Thus, thereis some possibility that studies of ribosomeorigins may eventually expand to include othercellular processes.

In the end, no matter how complete a pic-ture is developed of ribosomal developmentover time it will be hypothetical. The ultimateissue will be to prove at least the major partsof it. Thus, laboratory reconstructions willbe needed. However, there would be limitedvalue in resurrecting the complete ribosomeof LUCA, because it was in effect a modernribosome itself. An easier and likely equallyinformative task would be to obtain highresolution structural information on the mini-malized ribosomes found in various mitochon-dria. Laboratory reconstructions may insteadbest focus on examining meaningful pieces.

For example, in the case of both major tRNAsynthetase families, it is the catalytic subunitthat is by far the most conserved (O’Donoghueet al. 2003). Other less conserved subunits pro-vide the ability to recognize specific tRNAs andto edit charging errors. One can therefore infer atimeline for increased complexity of these mul-tisubunit enzymes in which the ability to ami-noacylate precedes these other features. That isto say, the ability to aminoacylate small RNAsmay predate the ability to distinguish individualRNAs as being appropriate targets for the addi-tion of particular amino acids. Thus, the firstsynthetases may have aminoacylated largelyrandomly. A relevant experiment then wouldbe to reconstruct an ancestral synthetase cata-lytic subunit and see if it can charge a onedomain tRNA and if so, with what amino acids.However, the critical first target for reconstruc-tion will be the PTC and efforts in this directionhave already begun (Davidovich et al. 2009). Afull fledged experimental program will becomepossible if it can be shown that a PTC fragmentcan catalyze peptide bond formation whenpresented with CCA terminated RNAs carryingamino acids.

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G.E. Fox




9, 20102010; doi: 10.1101/cshperspect.a003483 originally published online JuneCold Spring Harb Perspect Biol

George E. Fox Origin and Evolution of the Ribosome

Subject Collection The Origins of Life

Constructing Partial Models of Cells

et al.Norikazu Ichihashi, Tomoaki Matsuura, Hiroshi Kita,

The Hadean-Archaean EnvironmentNorman H. Sleep

RibonucleotidesJohn D. Sutherland

An Origin of Life on MarsChristopher P. McKay

Characterize Early Life on EarthHow a Tree Can Help−−Deep Phylogeny

RandallEric A. Gaucher, James T. Kratzer and Ryan N.

Primitive Genetic PolymersAaron E. Engelhart and Nicholas V. Hud

Medium to the Early EarthCosmic Carbon Chemistry: From the Interstellar

Pascale Ehrenfreund and Jan Cami

Membrane Transport in Primitive CellsSheref S. Mansy

Origin and Evolution of the RibosomeGeorge E. Fox

The Origins of Cellular LifeJason P. Schrum, Ting F. Zhu and Jack W. Szostak

BiomoleculesPlanetary Organic Chemistry and the Origins of

Kim, et al.Steven A. Benner, Hyo-Joong Kim, Myung-Jung

From Self-Assembled Vesicles to ProtocellsIrene A. Chen and Peter Walde

the Origins of LifeMineral Surfaces, Geochemical Complexities, and

Robert M. Hazen and Dimitri A. Sverjensky

The Origin of Biological HomochiralityDonna G. Blackmond

Historical Development of Origins ResearchAntonio Lazcano

Earth's Earliest AtmospheresKevin Zahnle, Laura Schaefer and Bruce Fegley

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

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