Gutell 083.jmb.2002.321.0215

Uploaded on


More in: Technology
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads


Total Views
On Slideshare
From Embeds
Number of Embeds



Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

    No notes for slide


  • 1. Modeling a Minimal Ribosome Based on ComparativeSequence AnalysisJason A. Mears1, Jamie J. Cannone2, Scott M. Stagg1, Robin R. Gutell2Rajendra K. Agrawal3,4and Stephen C. Harvey1*1Department of Biochemistryand Molecular GeneticsUniversity of Alabama atBirmingham, Birmingham, AL35295-0005, USA2Institute for Cellular andMolecular Biology and Sectionof Integrative BiologyUniversity of Texas at Austin2500 Speedway, Austin, TX78712-1095, USA3Wadsworth Center, New YorkState Department of HealthState University of New York atAlbany, Empire State PlazaP.O. Box 509, Albany, NY12201-0509, USA4Department of BiomedicalSciences, State University ofNew York at Albany, EmpireState Plaza, P.O. Box 509Albany, NY 12201-0509, USAWe have determined the three-dimensional organization of ribosomalRNAs and proteins essential for minimal ribosome function. Comparativesequence analysis identifies regions of the ribosome that have been evolu-tionarily conserved, and the spatial organization of conserved domains isdetermined by mapping these onto structures of the 30 S and 50 S sub-units determined by X-ray crystallography. Several functional domains ofthe ribosome are conserved in their three-dimensional organization inthe Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplastribosomes. In contrast, other regions from both subunits have shiftedtheir position in three-dimensional space during evolution, including theL11 binding domain and the a-sarcin–ricin loop (SRL). We examined con-served bridge interactions between the two ribosomal subunits, giving anindication of which contacts are more significant. The tRNA contacts thatare conserved were also determined, highlighting functional interactionsas the tRNA moves through the ribosome during protein synthesis. Toaugment these studies of a large collection of comparative structuralmodels sampled from all major branches on the phylogenetic tree, Caenor-habditis elegans mitochondrial rRNA is considered individually because itis among the smallest rRNA sequences known. The C. elegans model sup-ports the large collection of comparative structure models while providinginsight into the evolution of mitochondrial ribosomes.q 2002 Elsevier Science Ltd. All rights reservedKeywords: ribosome; conservation; rRNA; evolution; phylogenetics*Corresponding authorIntroductionThe ribosome is the site of protein synthesis inall living cells. It is composed of two subunits thatcombine to form a functional ribosome, which is a70 S particle in bacterial cells. In Escherichia coli,the smaller 30 S subunit is composed of the 16 SrRNA and 21 proteins, while the larger 50 S sub-unit contains 23 S and 5 S rRNAs along with 31proteins. It was long believed that ribosomal pro-teins were the principal catalysts for translation,but the discovery of catalytic RNAs1,2suggestedthat RNA might provide the catalytic sites. Thisidea was confirmed when the crystal structure ofthe 50 S subunit revealed an all-RNA peptidyltransferase site, indicating that the ribosome is aribozyme.3Proofreading of mRNA takes place atthe mRNA binding site on the 30 S subunit, andRNA is implicated in this process as well.4 – 6Thelarge subunit is monolithic and is believed to actas a relatively static platform, while the small sub-unit has been shown to be a dynamic structurethat moves during translation in a ratchet-likemanner.7,8While proofreading of the codon/anticodoninteraction and peptidyl transfer are the funda-mental processes in translation, several otherregions of the ribosome also have important func-tional roles in protein synthesis. Within the large0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reservedE-mail address of the corresponding author:harvey@neptune.cmc.uab.eduAbbreviations used: 3P, three phylogenetic domains;3P2O, three phylogenetic domains plus two organelles;MRP, mitochondrial ribosomal protein; mt-rRNA,mitochondrial ribosomal RNA; SRL, a-sarcin–ricin loop;CRW, comparative RNA web; cryo-EM, cryo-electronmicroscopy.doi:10.1016/S0022-2836(02)00568-5 available online at onBwJ. Mol. Biol. (2002) 321, 215–234
  • 2. subunit, the L11 protein and binding site of thetoxin a-sarcin/ricin, the a-sarcin–ricin loop (SRL;residues 2663–2777, E. coli numbering) arerequired for functional interactions with trans-lation cofactors, including elongation factors(EF-Tu and EF-G).9 – 11Within the 16 S rRNA the530 stem/loop and the 1490 region are involved inproofreading,12helix 27 (residues 885–912) hasbeen indicated as a conformational switch,13andseveral other areas are functionally significant fortranslation. The two ribosomal subunits must com-municate during proofreading, peptidyl transferand translocation, and bridge contacts betweenthe two subunits are important in this communi-cation, as are specific interactions between thetRNAs and the ribosome.Structural studies have begun to provideinsights into the details of various steps in thetranslation cycle. Crystal structures of individualribosomal subunits (50 S from an archeon,Haloarcula marismortui;1430 S from a thermophilicbacterium, Thermus thermophilus15,16) have beensolved at 2.4 to 3.3 A˚ resolution. The crystalstructure of the whole 70 S ribosome fromT. thermophilus was solved at 5.5 A˚ resolution.17Results from crystallography have been sup-plemented by cryo-electron microscopy (cryo-EM)images of the ribosome at various stages of thetranslation cycle.10,18– 20These advances allowexploration of structural relationships that areimportant for function. Here we examine whichregions of the ribosome have been most highlyconserved throughout evolution and define a“minimal ribosome” where structure–functionrelationships can be elicited.We have used comparative sequence analyses21,22to examine conservation of the various regions ofribosomal RNA through the Archaea, Bacteria andEucarya (herein named 3P, indicating three phylo-genetic domains) phylogenetic domains. An evenmore inclusive model was obtained by examiningconservation through these three phylogeneticdomains plus mitochondria and chloroplasts(herein named 3P2O, for three phylogeneticdomains plus two organelles). To investigate themost minimal, truncated ribosome, we haveexamined the rRNA sequence of C. elegansmitochondria.The work presented here suggests the minimalstructures required for translation, and it alsoaddresses how evolution affects interactions withinthe ribosome, and with translation cofactors. Pre-viously it was predicted that viewing ribosomalstructures from a phylogenetic perspective wouldaddress how rRNA structure evolves.23We findthat several functional domains are maintained asan RNA core in the reduced ribosomal models,but that sequence deletions and insertions requirethat the positions of some regions must havemoved in three-dimensional space duringevolution. The largest differences between widelydiverged species are at the periphery, away fromthe central core.ResultsConservation through three phylogeneticdomains (3P)The first step in building a minimal ribosome isto compare conservation through the three phylo-genetic domains: Archaea, Bacteria and Eucarya.There are five levels of conservation as defined atthe Comparative RNA Web (CRW) site†.24Positions that are present in more than 95% of thesequences are divided into four categories wherespecific nucleotides are conserved at that positionin (1) 99–100%, (2) 90–98%, (3) 80–89%, and (4)below 80% of the sequences. The fifth category isfor those positions that are not present in morethan 95% of the sequences. The results of thesecomparative studies can be viewed on the second-ary structure diagrams for both the 16 S rRNAand 23 S rRNA at the CRW site. The results wereused to generate the ribosome models shown inFigure 1(a) and (b). Nucleotides that are 99–100%conserved are called “universally conserved resi-dues”, and these are represented as red spheres inFigure 1. While 23% of the 16 S rRNA positionsare universally conserved (Table 1), the region ofgreatest conservation in the small subunit sur-rounds the area where the mRNA and tRNAsinteract and move through the ribosome (Figure1(a)). Other universally conserved areas are mostlyat the interface where the small subunit interactswith the large subunit, but the regions of domain Iinvolved in the formation of intersubunit bridgeshave the lowest percentage of universally con-served residues. The large subunit has a cluster ofuniversally conserved residues around the cleftwhere the acceptor stems of A and P-site tRNAsbind and peptidyl transfer occurs (Figure 1(b)).These regions are constructed from domains IVand V of the 23 S rRNA, and they exhibit thehighest percentage of universally conserved resi-dues (29 and 28%, respectively). Conservation alsoextends from the peptidyl transfer site to manyresidues associated with the polypeptide-conduct-ing tunnel.3,25,26The SRL is universally conserved,and the regions of rRNA that bind L1, L11 andother proteins contain many residues that areuniversally conserved. As is the case for the smallsubunit, many of the residues at the interface ofthe large subunit are universally conserved.Nucleotides that are 90–98% conserved arereferred to as “highly conserved residues”, andthey are represented by green spheres in Figure 1.In the small subunit, the highly conserved areasare generally located in the same regions as theuniversally conserved residues. They fill many ofthe gaps left in the sequence between universallyconserved nucleotides in the mRNA cleft and atthe interface side of the subunit. The platform ofthe small subunit is highly conserved, with a few† http://www.rna.icmb.utexas.edu216 Modeling a Minimal Ribosome
  • 3. universally conserved residues interspersed. Thisregion is involved in limited, specific interactionswith the E-site tRNA and mRNA, and the veryhigh level of conservation of these residuessuggests that they play additional important struc-tural and functional roles. Highly conserved resi-dues in the large subunit are concentrated nearthe universally conserved residues, but thepercentages for these residues are more equallydistributed throughout the six structural domains(Table 1).The third level of conservation for the threephylogenetic domains is the category we call “con-served positions”. These nucleotides are less than90% conserved at the nucleotide level but aremaintained as a position in at least 95% of thesequences in the three phylogenetic domain data-set (combining categories 3 and 4 from the CRWsite). This category represents the preservation ofspecific structural elements (e.g. base-pairs; stems;loops) at particular positions in the secondarystructure, even though specific nucleotides arenot required. These structural elements presum-ably act as scaffolds to give the more highly con-served residues critical orientations in three-dimensional space. These conserved positions arerepresented as dark gray helices in Figure 1. Theseresidues are most predominant in domains II andVI of the large subunit (60% of the sequence ineach).“Non-conserved regions” are those positionsthat are not present in 95% of the sequences in the3P dataset; these are represented as transparent,gray ribbons in Figure 1. In many cases, theseareas occur in the rRNA from some species butare completely absent from ribosomes in otherFigure 1. Conserved regions of the ribosome through three phylogenetic domains ((a) and (b)) and through threephylogenetic domains plus mitochondria and chloroplasts ((c) and (d)), mapped onto the small subunit ((a) and (c))and on the large subunit ((b) and (d)). Positions present in more than 95% of the sequences are shown in red, greenand gray. Red spheres represent those nucleotides that are universally conserved (99% conserved or greater), greenspheres designate highly conserved residues (90–98% conserved). Dark gray helices represent conserved positions(less than 90% conserved at the nucleotide level). The light, transparent gray ribbons represent regions that are notpresent in at least 95% of the sequences. Conserved proteins are shown in red and non-conserved are gray(summarized in Table 1). Main structural features for both subunits are indicated with the followingabbreviations: bk, beak; sh, shoulder; sp, spur; P, platform; L1, L1 arm; CP, central protuberance; L11, L11 arm; SRL,a-sarcin–ricin loop. The four models can be viewed from a 3608 perspective in movies at the Harvey laboratorywebsite ( a Minimal Ribosome 217
  • 4. species. Also, the majority of the insertions to theE. coli, T. thermophilus and H. marismortui rRNAsequences occur in these regions.27Non-conservedregions in the small subunit include helices 16 and17 (the shoulder, residues 404–499; Figure 1(a)),helices 33 and 33a (the beak, residues 1000–1040),and helix 6 (the spur, residues 79–100). The highestpercentages for non-conserved residues in the 23 SrRNA are found for domains I and III in thesecondary structure (Table 1), which are apparentlynot absolutely required. In general, non-conservedregions are located at the periphery of the smalland large subunits. The 5 S rRNA of the largesubunit is not included in this model. While thegeneral structure of the 5 S rRNA is conservedover three phylogenetic domains, the sequenceconservation is low.The number of sequences in the ribosomalprotein database is much smaller than for rRNA,so conclusions about their conservation are tenta-tive. Here we do not concern ourselves with thespecific conservation of amino acid residues or thepresence or absence of regions of the ribosomalproteins. Rather, we only consider whether thehomologous protein is present in the organisms inthe 3P dataset (see Materials and Methods). Withinthese limits, protein conservation is shown in Table2. Conserved proteins are shown in red, and non-conserved proteins are shown with transparentgray ribbons in Figure 1. As with rRNA, conservedproteins are generally located near the core of bothsubunits, and proteins that are not conserved tendto be located on the periphery of the two subunits.This finding indicates that several rRNA elementsthat interact with proteins are conserved in bothsubunits. A strong correlation exists between pro-tein conservation and RNA conservation withinthe structure.In summary, most of the 30 S and 50 S subunitsare conserved across the three phylogeneticdomains. Furthermore, the degree of conservationis highest near the functional centers of the twosubunits, as would be expected, and the com-ponents of the bridges between the two subunitstend to be conserved, particularly in the neighbor-hood of the active sites.Table 1. Comparative analysisCategory 3P conservation analysis (small subunit)All (1542)aI (568) II (352) III (476) IV (146)1 23 (351)b19 (106) 24 (84) 26 (126) 24 (35)2 10 (156) 9 (49) 11 (38) 11 (53) 11 (16)3 5 (80) 5 (27) 5 (17) 6 (29) 5 (7)4 35 (545) 37 (209) 39 (139) 28 (131) 45 (66)5 27 (410) 31 (177) 21 (74) 29 (137) 15 (22)Large subunitAll (2904) I (561) II (733) III (352) IV (396) V (587) VI (275)1 20 (595) 17 (95) 20 (147) 10 (35) 29 (113) 28 (167) 14 (38)2 12 (342) 12 (69) 13 (94) 12 (42) 9 (36) 12 (72) 11 (29)3 10 (282) 11 (63) 10 (73) 4 (15) 9 (36) 9 (54) 15 (41)4 41 (1201) 38 (214) 50 (363) 33 (116) 39 (155) 39 (229) 45 (124)5 17 (484) 21 (120) 8 (56) 41 (144) 14 (56) 11 (65) 16 (43)3P2O conservation analysis (small subunit)All (1542)aI (568) II (352) III (476) IV (146)1 12 (178) 9 (51) 12 (42) 13 (60) 17 (25)2 11 (175) 8 (44) 14 (51) 15 (70) 7 (10)3 7 (115) 5 (29) 9 (33) 9 (44) 6 (9)4 26 (395) 24 (137) 31 (109) 26 (122) 18 (27)5 44 (679) 54 (307) 33 (117) 38 (180) 51 (75)Large subunitAll (2904) I (561) II (733) III (352) IV (396) V (587) VI (275)1 5 (151) 1 (5) 3 (25) 0 (0) 12 (48) 11 (64) 3 (9)2 7 (202) 6 (32) 8 (48) 0 (0) 11 (45) 9 (50) 6 (17)3 6 (168) 2 (9) 6 (47) 0 (0) 8 (33) 9 (52) 10 (27)4 22 (646) 11 (61) 31 (224) 0 (0) 29 (113) 28 (163) 31 (85)5 60 (1737) 81 (454) 52 (379) 100 (352) 40 (157) 44 (258) 50 (137)There are five categories listed; the first four include positions present more than 95% of the time: (1) 99–100% nucleotide conserva-tion, (2) 90–98% nucleotide conservation, (3) 80–89% nucleotide conservation, (4) less than 80% nucleotide conservation, (5) positionspresent less than 95%.aComparative results are shown by domain (All ¼ all domains, I ¼ domain I, etc.) with the total number of residues in that domainlisted in parentheses.bPercentages are listed (number/total) with the observed number of residues shown in parentheses. Additional comparative infor-mation is available at the CRW site ( Modeling a Minimal Ribosome
  • 5. Conservation through three phylogeneticdomains plus two organelles (3P2O)Due to the additional sequence and structurevariation in the mitochondria and to a lesser extentin chloroplast ribosomes, there is less conservationwhen these sequences are added to the 3P dataset.Figure 1(c) and (d) shows conservation across thethree phylogenetic domains plus the twoorganelles, using the same coloring scheme as inFigure 1(a) and (b) (3P2O secondary structures areavailable for both the 16 S and 23 S rRNA at theCRW site†).The small subunit model for 3P2O conservation(Figure 1(c)) is similar to the model based on 3Pconservation (Figure 1(a)), although overall con-servation is significantly lower. Many of the 3Puniversally conserved residues are only highlyconserved in the 3P2O model. Roughly half of theuniversally conserved residues fall to a lowerdegree of conservation for each domain (Table 1).The head and platform portions of the subunit inthe 3P2O model are very highly conserved, andmost of the universally conserved residues arefound at or near the mRNA binding site. Feweruniversally and highly conserved residues arefound in the lower body regions, except for someat the interface. Although overall conservation inthe body is not as high as in the 3P model, 29% ofthe residues in domain I are conserved as posi-tions, indicating that the structural elements arestill required at these positions.The number of universally conserved residues isdramatically reduced in the 3P2O model for thelarge subunit (constituting only 5% of thesequence, Table 1), and almost all of the peripheralrRNA is non-conserved (60% of the positions arenot maintained in 95% of the sequences in theTable 2. Protein conservationSSU protein 3P 3P2O In crystal?aLSU protein 3P 3P2O In crystal?aS1 2 2 NobL1P þ þ NocS2 þ þ Yes L2P þ þ YesS3 þ þ Yes L3P þ 2 YesS4 þ þ Yes L4P þ 2 YesS5 þ þ Yes L5P þ þ YesS6 2 2 Yes L6P þ 2 YesS7 þ þ Yes L7AE þ 2 YesS8 þ þ Yes LPO (L10P) þ 2 NoS9 þ þ Yes L10E (L10AE) þ þ YesS10 þ þ Yes L11P þ þ NocS11 þ þ Yes L12A (L7/L12) þ 2 NoS12 þ þ Yes L13Pdþ 2 YesS13 þ þ Yes L14Ed2 2 NoS14 þ 2 Yes L14P þ 2 YesS15 þ 2 Yes L15E 2 2 YesS16 2 2 Yes L15P þ 2 YesS17 þ þ Yes L18E 2 2 YesS18 2 2 Yes L18P þ þ YesS19 þ þ Yes L19E 2 2 YesS20P 2 2 Yes L21E 2 2 YesS21 2 2 NoeL22P þ þ YesS22 2 2 No L23P þ þ YesLTRA 2 2 No L24E 2 2 YesTHX 2 2 Yes L24P þ þ YesL29P þ 2 YesL30E 2 2 NoL30P þ 2 YesL31E 2 2 YesL32E 2 2 YesL34E 2 2 NoL35AE 2 2 NoL37E 2 2 YesL37AE 2 2 YesL39E 2 2 YesL40E 2 2 YesL41E 2 2 NoL44E 2 2 YesLX 2 2 NoConservation based on website crystal structures are a bacteria (T. thermophilus, 1FJG) for the SSU and an archea (H. marismortui, 1FFK) for the LSU.bS1 was removed from the 30 S subunit before crystallization.cProteins L1P and L11P were modeled onto the structures for spatial representation.dP and E represent prokaryotic and eucaryotic ribosomal proteins, respectively.eS21 is not found in T. thermophilus.† http://www.rna.icmb.utexas.eduModeling a Minimal Ribosome 219
  • 6. 3P2O dataset). Much of the reduction is attribu-table to animal mitochondrial ribosomes, and notto plastid (chloroplast) ribosomes because theirconservation is similar to the bacterial 70 Sribosomes.28,29A universally conserved core stillremains at the cleft near the peptidyl transferasesite (domains IV and V), but most of the polypep-tide-conducting tunnel is absent (Figure 2). Highlyconserved residues or conserved positions makeup most of the remaining core. The SRL is still uni-versally conserved, indicating its essential role.Some universally conserved residues are found atthe interface, but many of the subunit–subunitbridge contacts are apparently lost in organellarribosomes, as is discussed in more detail below.The 5 S rRNA is not included in the 3P2O model,since the majority of the mitochondria do not havea 5 S rRNA. All organisms that have the 5 S rRNAalso contain the 2282–2394 region of the 23 SrRNA that is protected by 5 S rRNA.While the majority of the deletions relative to theE. coli, T. thermophilus and H. marismortui secondarystructures are on the periphery of the rRNA three-dimensional structure, a few are internal withislands of conservation at the outer edges of the3P2O structural model. One example is the L11protein binding domain (residues 1055–1105).This conserved region is located at the peripheryof the subunit and interacts with translationcofactors, like EF-G.30The nearest connecting helixto the conserved L11 binding domain is roughly80 A˚ away in the 3P2O model. This raises thepossibility that the position of the rRNA hasmoved during evolution. The distance from theL11 binding domain to the central core of the par-ticle has to be smaller, as the adjoining helix (helix41 and/or 42) is absent from the 3P2O model. TheSRL is also on the periphery of the subunit, andits position must have also evolved to accommo-date changes in the size of the ribosome. Both theSRL and the L11 binding domain are important inbinding elongation factors, so their positions musthave evolved together. Another interesting featureof the 3P2O model is that protein L1 is conserved,but the rRNA that it contacts in cytoplasmic ribo-somes (residues 2109–2180) is not conserved inorganellar ribosomes. Recent studies raise thepossibility of structural compensation by ribo-somal proteins for loss of parts of the rRNA withinbovine mitochondrial ribosomes.31 –34However, it isnot clear which protein would be involved in eachcompensation.The most dramatic loss of RNA using 3P2O con-servation analysis in the 23 S secondary structureFigure 2. The polypeptide-con-ducting tunnel is illustrated forboth (a) 3P and (b) 3P2O conserva-tion by taking a cross-section of the50 S subunit. The coloring schemeis the same as in Figure 1, with theexception that regions that are notpresent in at least 95% of thesequences are not transparent, butthey are still colored light gray. TheP-site tRNA and an elongated poly-peptide in the tunnel are coloredblue. The tunnel is conservedthrough three phylogeneticdomains, but it is severely trun-cated when mitochondria andchloroplast sequences are included.220 Modeling a Minimal Ribosome
  • 7. occurs in domain III (100% non-conserved; Table 1).It may be that this region is not essential fortranslation, but that it has been developed tomake the process more efficient or to assist withcomplementary functions (i.e. protein secretion).Also, most of domain I is absent in 3P2O conserva-tion (81% non-conserved), and the few RNAloops that are conserved are close to the conservedcore.Few proteins are shown in Figure 1(c) and (d),but it should be noted that mitochondrial ribo-somes contain more proteins than their bacterialcounterparts. Unfortunately, the limited knowl-edge about mitochondrial ribosomal proteins(MRPs) makes it difficult to say exactly whichproteins are conserved and how they are arrangedin three-dimensional space (see Materials andMethods).A model for the C. elegansmitochondrial ribosomeWhile these comparative phylogenetic studieshave suggested a model of the minimal ribosome,we also developed a model of an extremelyreduced ribosome found in nature for comparison.The rRNA from C. elegans mitochondria wasmodeled, since this ribosome contains one of themost reduced small and large subunit sequencesknown.35Figure 3 shows the alignment of thesesequences with the sequences of the species usedin the crystal structures (T. thermophilus for thesmall subunit and H. marismortui for the large sub-unit). This gives the information needed to gener-ate the corresponding three-dimensional models(Figure 4). The MRPs found in C. elegans are notwell defined, so we have not taken them into con-sideration. Nucleotides that align between theC. elegans mitochondrion and the species from thecrystal structures are shown in red in Figures 3and 4. Yellow indicates regions where the C. eleganssequence does not align with that from the speciesin the crystal structures. The secondary structureof the yellow regions has not been determined, inpart because the C. elegans sequence is very A-Urich. They are mapped to the corresponding regionof the three-dimensional structure even though theactual structure of the C. elegans rRNA may differsubstantially from that in the crystal structures.However, since these unassigned sequences flankconserved regions, a rough estimate of where theRNA sequence would be located in the model canbe made. It should be pointed out here again thatthe 5 S rRNA has not been found in animal mito-chondrial genomes, including C. elegans.The models of the C. elegans mitochondrial ribo-somal RNAs (mt-rRNAs) are very similar to thoseobtained using 3P2O phylogenetic conservation.The small subunit mt-rRNA maps to the regionaround the head and mRNA binding site of thebacterial small subunit. The platform is also con-served in this model, but most of the rRNA in thebody is lost. Missing regions in the small subunitinclude the beak, helices 16 and 17, and most ofthe periphery. Certain regions at the interface areconserved, including the uppermost region ofhelix 44 (residues 1401–1415; 1485–1501). Thelarge subunit also looks very similar to the 3P2Omodel. A core region around the peptidyl transfer-ase site and the interface is conserved. The con-served regions at the interface of the large subunitare complementary to those that they interact within the small subunit. In particular, helices 69 and71 (residues 1906–1961 in E. coli) are conserved.These helices are known to interact with helix 44of the small mt-rRNA. It should be noted that theunaligned regions of the C. elegans sequence(yellow) are located near the periphery of theconserved core, and both the L11 protein-bindingand SRL domains are present.As with the 3P2O model, the principal deletionsin sequence occur mainly in the periphery of thesmall and large subunits. However, a number ofexceptions are listed in Table 3, along with ananalysis of the effects of losing these sequences onthe three-dimensional structure of C. elegans mt-rRNA. The distances between consecutive nucleo-tides on either side of the deletion are reported,and any unaligned sequence that fits in the gap ofthe deletion is listed in the next column. On thebasis of the distance to be spanned and the lengthof the unaligned sequence, we have estimated theprobability, P(move), that the regions on eitherside of the gap must be moved closer together inorder to permit closure of the RNA chain. Briefly,for a given gap length of N unaligned nucleotides,we built a histogram of end-to-end distances forall N-nucleotide fragments within the crystallo-graphic database. If the distance between the lastphosphate before the gap and the first phosphateafter the gap in the model is r, then the probability,P(move), that such a gap cannot be closed by afragment with N nucleotides using a structuralmotif resembling one found in the crystallographicdatabase is simply that fraction of the histogramdistances that is less than r (see Materials andMethods for details). In this way it is possible tosee which conserved regions in the C. elegans sub-units probably have different positions in three-dimensional space than the corresponding regionsdo in the available crystal structures.Several loops in the small subunit align toregions far from their respective helices. Anexample of this is the loop at the end of the penul-timate stem, or helix 44 (residues 1450–1452)where three nucleotides align. They are roughly80 A˚ away from the nearest connecting helix, sothe position of this loop in three-dimensionalspace must be different from that in bacteria. Ofthe deletions that are found in the small subunit(Table 3), all but two result in some conservedfeature being in a position where it cannot reachthe nearest connecting helix. This suggests thatthese regions must have moved during evolution.One exception is the deletion of helices 16 and 17where three unaligned nucleotides are available toModeling a Minimal Ribosome 221
  • 8. Figure 3 (Legend opposite)
  • 9. Figure 3. The alignment of C. elegans mitochondrial (a) small and (b) large rRNA to the secondary structures ofT. thermophilus and H. marismortui, respectively. Red residues represent nucleotides that align, while yellow residuesrepresent nucleotides that are found in C. elegans but could not be aligned with the correlating secondary structure.Since these unassigned sequences flank conserved regions, a rough estimate of where the RNA sequence would belocated in the three-dimensional model (Figure 4) can be made, but the structure may be different from the secondarystructure, since these sequences do not align. A few functional regions are labeled and helix numbers are shown ingreen for those areas that are mentioned in the text.Modeling a Minimal Ribosome 223
  • 10. Figure 4. Mapping the alignment of C. elegans mitochondrial rRNA to the crystal structures of T. thermophilus (1FFK)and H. marismortui (1FJG). The red helices represent those nucleotides that do align. The yellow helices are used forspatial representation of C. elegans sequence that does not align, but is inferred to be in a particular region of thethree-dimensional structure. Mitochondrial proteins are not included. Both models can be viewed from a 3608 perspec-tive in movies at the Harvey laboratory website ( 3. Deletions in the C. elegans alignmentSmall subunitHelices losta,bDeletioncGap distance (A˚ ) Sequence length (nt)dP(Move)eSH5–15 51–393 Loopf44 –SH16–17 408–499 17 3 0.3SH21–22 (690 region) 588–672, 736–760 65, 43 None 1.0SH25–26a (3 nt loop) 814–863, 867–879 35, 36 None 1.0SH33 and SH35 996–1045 16 NA 1.0SH37 (6 nt loop) 1084–1089, 1096–1102 25, 17 None 1.0SH38–40 1113–1191 23 11 0.2SH41 (3 nt loop) 1243–1265, 1269–1294 35, 29 None 1.0LH44 (3 nt loop) 1416–1449, 1453–1484 83, 97 None 1.0Anti-Shine-Delgarno 1536–1542 ? None –Large subunitLH1–25 1–562 NA 22 (50end) –LH28–31 595–662 Loopf4 –LH34 (3 nt loop) 698–713, 717–763 26, 26 None 1.0LH37–42 and LH45 (GTPase region) 818–1050, 1109–1185 91, 100 35, 13 1.0LH46 (4 nt loop) 1206–1222, 1227–1240 36, 19 None 1.0LH47–63 1276–1765, 1987–2010 65, 46 44, 19 0.7LH66 (4 nt loop) 1798–1806, 1811–1821 26, 30 None 1.0LH68 (3 nt loop) 1844–1868, 1872–1896 51, 60 None 1.0LH76–79 2092–2227 Loopf4 –LH81–88 2259–2421 26 24 0.2LH94 (connects to SRL) 2627–2645 43 8 1.0LH96 2679–2728 Loopf5 –LH97 2745–2759 LoopfNone –LH98–101 2770–2904 NA None (30end) –Deletions of 5 nt or less are not included.aSH and LH are used to represent small subunit helices and large subunit helices, respectively.bThe identity shown in red is the region that would need to move to compensate for the deletion.cThe residues that are deleted from the C. elegans sequence are listed with E. coli numbering.dSequence length represents the unassigned sequence from the C. elegans structure that is not represented by a homologous regionin the E. coli secondary structure and is therefore available to close the gap.eProbability that movement would be required to compensate for the deletion (see the text for explanation).fGap distance is not important because the deletion is at the end of a helix and can easily be closed by a new loop.224 Modeling a Minimal Ribosome
  • 11. cover a gap distance of 17 A˚ . The probability ofmovement is only 30%, indicating that thisdistance can be closed by the unaligned nucleo-tides. The 690 region, which has been implicatedin interactions with the P and E-site tRNAs in thesmall subunit,36,37must have moved during mito-chondrial evolution because there is a gap of 65 A˚on the Watson strand and 43 A˚ on the Crick strandof the deleted sequence. No unaligned nucleotidesare present to span this distance, so the probabilityof movement is 100%.The large subunit also has several deletions,resulting in large gaps. Some examples of deletionsdo not require movement because of unalignedsequence. The region in domain V that interactswith the 5 S rRNA (helices 81–88, residues 2259–2421) is absent, as was found in 3P2O conservation.In C. elegans there are 24 unaligned nucleotidesin this region. Such a large fragment could easilyclose the remaining 26 A˚ gap, and the probabilitythat movement was required during evolution iscalculated at only 19.5% (Figure 5).The large subunit also has regions that musthave moved during evolution to close gaps left bydeletions. These movements also correlate to theresults found in the model of 3P2O phylogeneticconservation. The L11 binding domain (helices 43and 44) is found in C. elegans, but the connectinghelices (41 and 42) are shortened. A total of 48nucleotides of unaligned sequence are in thisregion, but the distance of 91 A˚ would be difficult tospan with 35 nucleotides on the Watson strandof the helix, and 13 nucleotides cannot span 100 A˚on the Crick strand of the helix. We therefore con-clude that the position of the L11 binding domainmust have moved during evolution. Similarly, theSRL (at the end of helix 95) must have moved,because the deletion of helix 94 and the base ofhelix 95 creates a gap of 43 A˚ , and the eightunaligned nucleotides in this region cannot spanthat distance (Figure 5). Since both the SRL andthe L11 binding domain are known to interactwith translation cofactors on the surface of theribosome, comparison of C. elegans withH. marismortui reveals substantial differences inthose interactions. At present, it is unclear whetherthe interactions between cofactors and the largesubunit are simply moved to a different locationin the C. elegans large subunit (but are otherwisesimilar to those in H. marismortui), or whetherother parts of the subunit (such as proteins notfound in H. marismortui) take on roles otherwiseassociated with the SRL, L11 and the L11 bindingdomain. The latter appears unlikely because thesespecific regions are so highly conserved.Conservation of intersubunit bridgesThe two ribosomal subunits must communicatewith one another to synchronize various eventsduring translation. This communication takesplace at the interface of the subunits through themovements of bridges. These bridges have beenshown to be dynamic, with different connectionsmade and broken at different stages of the trans-lation cycle.8,38Table 4 examines the conservationof intersubunit bridges, including the type of inter-action (RNA/RNA, RNA/protein, protein/pro-tein), whether or not the bridge is conservedbased on 3P and 3P2O conservation, and if therRNA sequence involved can be inferred to existin C. elegans. At the 3P conservation level, all 11listed bridges are conserved. This points to theimportance of these bridges for both the structuralintegrity of the ribosome and the probable func-tional significance of these interactions intranslation.However, the picture is notably different whenmitochondria and chloroplasts are included,because only four of the 11 bridges are conservedacross 3P2O (Table 4). Bridges B2a, B2c, B3 andB7b are conserved, indicating that these contactsare probably essential. B2a and B3 involve contactsnear the decoding site at the top of the penultimatestem in the small subunit. From cryo-EM and crys-tallography studies, it has been shown that thispart of helix 44 moves during translation,4,8,39andthese bridges almost certainly play a role in thosemotions.Bridge B2c is also found near the decoding siteand is conserved through all three categories listedin Table 4. This bridge involves helix 27 in thesmall subunit, often referred to as a “conformationalswitch” because it has been shown to adopt twosecondary structure conformations. Mutations thatprevent conformational changes in this regionaffect fidelity.13,40This switch lies adjacent to helix44, which, as pointed out above, changes confor-mation during the translation cycle. Fidelity mayinvolve the coordination of the movement of thehelix 27 switch with that of the decoding site inhelix 44. Bridge B2c also includes contacts withthe highly conserved structure, helix 24 in thesmall subunit, the tip of which contains the 790loop, known to be involved in interactions withthe mRNA and tRNA at the ribosomal P-site.41,42In general, bridges that are positioned furtherfrom the decoding site tend not to be conserved inthe 3P2O model (Figure 6). Bridges B5 and B6involve contacts between the large subunit andthe penultimate stem of helix 44 in the small sub-unit, but they are further from the decoding sitethan B2a and B3 and are not conserved in the3P2O model. Another example is bridge B1a,formed by helix 38 of the large subunit and oftenreferred to as the “A-site finger”. This structure isnot conserved in mitochondria, which correlateswith the loss of much of the rRNA in the centralprotuberance, and with the loss of 5 S rRNA inmitochondria. Similarly, B7a is not conserved, asthis bridge is positioned away from the decodingsite and closer to the L1 arm of the large subunit,connecting to the platform of the small subunit.Bridges B4, B7a and B8 are located further awayfrom the central region of both subunits, and theyare not conserved.Modeling a Minimal Ribosome 225
  • 12. Figure 5. Probability of movement. (a) The absence of helix 94 (residues 2630–2643; 2761–2777) from the C. eleganslarge ribosomal subunit leaves only eight unaligned nucleotides to span a distance of 43 A˚ if the neighboring helicesare to occupy the same positions as in the H. marismortui crystal structure. The histogram shows the inter-phosphatedistance distribution for all nine-nucleotide fragments in the 70 S crystal structures (1GIX and 1GIY). Since 99.3% ofsuch fragments are too short to span a 43 A˚ gap, we estimate that there is a 99.3% probability that helices on one orboth sides of the gap must occupy different positions in the C. elegans structure than in the H. marismortui structure.(b) Absence of helices 81–88 (residues 2259–2421) from the C. elegans large ribosomal subunit leaves a gap of 26 A˚ tobe spanned by 24 unaligned nucleotides. In all, 80.5% of all 25-nucleotide fragments in structures 1GIX and 1GIYspan distances greater than this, so the probability that some part of the C. elegans rRNA must be moved to close thegap is only 19.5%.226 Modeling a Minimal Ribosome
  • 13. Table 4. Bridges conserved through evolutionBridgeaSSU positionsbLSU positionsb3Phylo3 Phylo þ 2Org C. elegans Bridge typeB1a S13 (92–94) H38 (886–888) Yes No No RNA/pro-teinB2a H44 (1408–1410) H69 (1913–1914, 1918) Yes Yes Yes RNA/RNAB2b H24 (784–785, 794), H45 (1516–1519) H67 (1836–1837, 1922), H71 (1919–1920, 1932) Yes No No RNA/RNAB2c H24 (770–771), H27 (900–901) H67 (1832–1833), H67 (1832–1833) Yes Yes Yes RNA/RNAB3 H44 (1484–1486) H71 (1947–1948, 1960–1961) Yes Yes Yes RNA/RNAB4 H20 (763–764), S15 (40–44) H34 (717–718), H34 (713, 717) Yes No No RNA/pro-teinB5 H44 (1418–1419), H44 (1420–1422), H44 (1474–1476), H44 (1474–1476)H64 (1768–1769), L14 (44–49), H62 (1689–1690), H64(1989)Yes No No RNA/pro-teinB6 H44 (1429–1430, 1474–1476), H44 (1431) H62 (1689–1690, 1702–1705), L19 (Hm24e:R44) Yes No No RNA/pro-teinB7a H23 (698, 702) H68 (1848–1849, 1896) Yes No No RNA/RNAB7b H23 (712–713), H24 (773–776) L2 (162–164, 172–174, 177–178), L2 (177–178, 198–202) Yes Yes ?cRNA/pro-teinB8 H14 (345–347) L14 (116–119) Yes No No RNA/RNAThe positions are based on the crystal structure of the E. coli 70 S ribosome (Yusupov et al.17, 1GIX and 1GIY).aB1b not included because the bridge type is protein/protein.bSmall subunit (SSU) and large subunit (LSU) positions are listed by helix (H) or protein (S or L) using E. coli numbering.cThe rRNA for B7b is conserved, but it is not known whether or not protein L2 is present. Therefore, we cannot say whether or not this contact exists in C. elegans.
  • 14. Although it is not found in the core of the ribo-some, bridge B7b is conserved at the 3P2O level.B7b arises from an interaction between protein L2and helix 23 from the small subunit. L2 is highlyconserved in all organisms and organelles, as ishelix 23. The conservation of B7b is less clear inthe C. elegans mitochondrial ribosome, because L2has not been found in C. elegans, and half of thenucleotides involved in helix 24 (residues 712–713) are lost. If C. elegans contains a bridge corre-sponding to B7b, the details of the intersubunitinteractions must be different than in other ribo-somes. We indicate the uncertainty about the exist-ence of bridge B7b by coloring it yellow in Figure 6.Conservation of tRNA/rRNA contacts inthe ribosomeMany tRNA interactions with the ribosome aremade and severely broken throughout the trans-lation cycle as the tRNA moves through the centralcavity, the intersubunit space, between the ribo-somal subunits. The large subunit interacts withthe acceptor stem and elbow region of the tRNA,while the small subunit interacts with the anti-codon stem and loop. Table 5 lists the tRNA inter-actions with the ribosome and the residuenumbers that are involved in the contacts, basedon the recent T. thermophilus 70 S crystal structure.17A distance criterion (8.5 to 10 A˚ ) was also used todetermine residues near the different tRNA posi-tions and mRNA. While this list is more inclusive,the results are similar to those shown in Table 5(additional Tables are available at the CRW site†).On the basis of the crystal structure, the A-sitetRNA has few interactions with the small subunit,but these are vital to the fidelity of translation.Residues 530 and 1492–1493 are believed to readthe groove of the mini-helix created by thecodon–anticodon interaction at the A-site,4and allthree of these residues are universally conservedin all organisms and organelles. Residues 955 and1054 are also conserved, but only at the level ofconserved positions in both the 3P and 3P2Omodels, indicating that these residues may beimportant in positioning the tRNA but are prob-ably not involved in specific hydrogen bonding.Several interactions occur between the A-sitetRNA and the large subunit. The greatest degreeof conservation is found in the residues that con-tact the tRNA acceptor stem. Residues in the loopbetween helices 69 and 71 and other nucleotidesthroughout domain V position the acceptor stem.Conserved residues within helix 89 and the loopof helix 69 interact with the elbow region of theA-site tRNA in the 3P2O model, but residueswithin the A-site finger (helix 38; residues 880–900) that would normally contact the elbow of thetRNA are not conserved in some mitochondria.Similarly, residues in domain I of the large subunitthat contact the elbow of the A-site tRNA in the3P model are missing from the 3P2O model. Inshort, ribosome contacts with the acceptor stem andanticodon at the A-site are most highly conserved,while interactions with the elbow region are con-served in the 3P model, but not in the 3P2O model.Figure 6. Positioning of bridge contacts onto densitymaps of the large subunit (upper) and small subunit(lower). Red bridges (B2a, B2c and B3) represent posi-tions that are present in at least 95% of the sequencesthrough 3P and 3P2O conservation and are also foundin C. elegans. B7b is colored yellow because it is con-served through 3P and 3P2O, but it cannot be deter-mined if it is present in C. elegans (it is not knownwhether protein L2 is conserved in C. elegans). The otherseven bridges are conserved through 3P, but are not con-served when organelles are considered, and they are alsoabsent from C. elegans mitochondrial rRNA (Table 3).† Modeling a Minimal Ribosome
  • 15. The P-site tRNA is at a crucial position in theribosome because it carries the nascent peptidechain. Several tRNA/rRNA contacts are madewith both subunits, and all of these interactionsare conserved through both the 3P and 3P2Olevel. Within the small subunit, C1400 and residues1338–1339 are universally conserved. Universallyconserved residues in the large subunit includeresidue 2252 in the P-loop and 2602 in helix 93,which appear to be crucial for positioning theacceptor stem in the P-site. Other large subunitcontacts with the acceptor stem and with theelbow of the tRNA are conserved as positions, butnot as specific residues, again indicating that theirrole is probably to help position the tRNAs, forwhich a specific hydrogen bonding pattern isapparently not needed. It is impressive that all ofthe contacts between the P-site tRNA and therRNA are conserved at some level, reflecting theimportance of the positioning of the tRNA at thisposition.The E-site tRNA also has contacts with both sub-units and, like the A and P-site tRNAs, all of itsinteractions with the small subunit are conservedat some level. Residue 1382, found in helix 28 ofthe small subunit rRNA, is universally conserved.Nucleotides in the 690 loop and 790 loop are uni-versally conserved throughout 3P2O, but othernucleotides that contact the E-site anticodon areonly conserved as positions. Ribosomal nucleotidesin the large subunit that interact with the E-sitetRNA are conserved at the 3P level, but only aspositions, indicating a role in positioning thetRNAs. Interestingly, most of the E-site contactswithin the large subunit are lost when mito-chondrial sequences are considered, raising thequestion of whether or not a true E-site exists inall mitochondria. Only residues 2235 and 2433–2434, located in helix 75, are conserved throughout3P2O, including C. elegans. Residue 199 is found in3P2O conservation but is missing from C. elegans.All of these residues contact the acceptor stem ofthe E-site tRNA.Discussion and ConclusionsWe have used comparative sequence analysis toexamine conserved features of ribosome structure.Most of the rRNA and proteins in the 70 S particleare conserved to some degree across the 3Pdomains. The regions that are not conserved at the3P level tend to be located on the periphery of thestructure. These reflect the fact that Archea,Bacteria and Eucarya ribosomes vary in size, andthe likelihood that organisms living in widelydifferent environments have developed differentribosomal modifications to guarantee translationalaccuracy and efficiency. The universally conservedresidues are generally found in areas that areknown to be functionally significant, particularlynear the mRNA binding site on the small subunit,the peptidyl transferase site on the largeTable 5. Conserved tRNA/rRNA contacts within theribosome. (More quantitative details available at theCRW site ( numbers tRNA residues 3P 3P2O C. elegansSmall subunit5301 34–36 Yes Yes Yes955 40 YesaYesaYes1054 34 Yes YesaYes1493 38 Yes Yes YesLarge subunit27,30 55,62 YesbNo No881–2 17 YesbNo No882–3 19 YesbNo No898–9 56 No No No1913–4 25,26 Yes YesaYesc1914–5 11,12 Yes YesaYesc1942–3 72–73 Yes Yesa,cYes2452 and 2494 74–76 Yes Yes Yes2470–2 50–53 YesbYesbYes2482–4 64–65 YesbYesbYesc2553 75 Yes Yes YesP-SiteSmall subunit790 38 Yes Yes Yes966 34 YesaYesaYes1229 28–30 YesaYesaYes1338 41 Yes Yes Yes1339 40 Yes Yes Yes1400 34 Yes Yes YesLarge subunit1908–9 12, 13 YesaYesaYes1922–3 25, 26 Yes YesaYes2252 74 Yes Yes Yes2255–6 3 Yes YesbYes2585 76 Yes YesaYes2602 75 Yes Yes YesE-SiteSmall subunit693–695 37–9 Yes YesbYes788–789 37–8 YesbYesaYes937 33 YesaYesaYes1339–40 35–6 YesbYesbYes1382 34 Yes Yes YesLarge subunit199 76 Yes Yes No1850–3 3, 4, 5 YesbNo No1852–3 2,71 YesbNo No1892 71 Yes No No2112–3 19 YesaNo No2116–7 56 YesaNo No2235 73 YesaYesaYes2433–4 76 Yes YesbYesThe residues listed are based on the 70 S crystal structure17and are listed with E. coli numbering.aResidue(s) position(s) conserved in at least 95% of thesequences, but conservation is not greater than 90% at thenucleotide level.bResidue(s) universally or highly conserved, but one of theresidues is conserved as a position (less than 90% at the nucleo-tide. level, but position is present in at least 95% of thesequences).cOne of the residues in this region is not conserved.Modeling a Minimal Ribosome 229
  • 16. subunit, and the regions on the large subunit thatinteract with elongation factors. A strong corre-lation also exists between protein conservationand conservation of regions of the rRNA that inter-act with those proteins. While this may seem anatural consequence of the need to preserveprotein-binding sites, the situation becomes quitedifferent when mitochondria and chloroplasts aretaken into consideration.As seen in Figure 1(c) and (d), when organellesequences are considered, both subunits showsevere truncations. Even though plastid ribosomesare very eubacterial-like, the minimal modelsbased on 3P2O conservation are quite simple,which reflects the diversity of mitochondrialsequences, and the simplicity of many of them.The simplification of mitochondrial rRNAsequences correlates well with the known simpli-fication of mitochondrial genomes. Mitochondriaimport many essential proteins and RNAs.Although it is not yet fully understood howevolution has chosen which protein and RNAgenes to move from the mitochondrion to thenucleus, natural selection must balance efficiencyagainst the need to regulate levels of geneexpression. The diversity and simplicity of mito-chondrial genomes must reflect these competingdemands. One interesting facet of this is theobservation that some mitochondrial ribosomalproteins have C and N-terminal extensions,apparently to accommodate for the loss of rRNA.34This explains the increased ratio of protein toRNA within mitochondrial ribosomes. As moredata on MRPs become available, it will be interest-ing to understand how proteins replace rRNAboth structurally and functionally.Those proteins that maintain conserved inter-actions with rRNA are generally known to servecritical roles in translation. Roughly half of thesmall subunit proteins (13/24) are conservedacross all three phylogenetic domains and bothorganelles (Table 2), indicating their importance inthe structure and function of the small subunit.For example, proteins S5 and S12 are conserved,as is helix S27 with which they interact. Mutationalstudies have shown the importance of theseproteins in translational fidelity.13Although all of the bridges are conserved acrossthe three phylogenetic domains, only B2a, B2c, B3and B7b are conserved across 3P2O (Table 4),suggesting that these are ancient and functionallycritical contacts. The functional significance of B2aand B3 has been suggested by several studies, but,as far as we are aware, the importance of B2c andB7b has not been previously suggested. Proteinsmay replace RNA in some of the bridges that arenot conserved across 3P2O, but the reduced sizeof both subunits in the 3P2O consensus modelsindicates that other bridges, particularly thosearound the periphery, do not exist in all ribosomes.The disconnected appearance of the 3P2Omodels in Figure 1 arises from the lack of conserva-tion of critical RNA connections, suggesting thatsome functional regions have moved duringevolution. For example, L11 and the rRNA thatbinds it are both conserved across 3P2O, while theadjoining helices (helices 37–42 and helix 45) arenot. Similarly, helix 94, which connects the SRL tothe rest of the ribosomal RNA, is missing in the3P2O consensus model. The remaining rRNA frag-ments that must connect the SRL and the L11region to the rest of the large subunit are too shortto do so without some change in the positions ofthe SRL and protein L11 (Table 3). Therefore, theSRL and L11 must both be in substantially differentpositions in mitochondrial ribosomes than in otherribosomes. Similarly, the 690 region of the smallsubunit rRNA is separated from its neighboringsequence in the 3P2O model, because of the lossof the S15 protein-binding region. Theseobservations strongly suggest that, as pieces ofrRNA are added or deleted during evolution, therelative three-dimensional positions of some highlyconserved RNA domains must change. Theseevolutionary shifts may well alter both intra-subunit and intersubunit interactions. The func-tional consequences of such changes are unclear.The polypeptide-conducting tunnel is conservedin the 3P model, but the truncation of rRNA in the3P2O model results in a very short tunnel (Figure2). It is likely that as the need for protein secretionarose, the protein tunnel evolved, which is whythis region is so highly conserved in the 3P model.The truncation of the tunnel in many mito-chondrial sequences is surprising, since manyproteins translated in the mitochondria aremembrane-associated. The strategy for proteintranslocation is conceivably very different inorganelles, so that mitochondrial ribosomes havedeveloped a new mechanism for protein secretion.The non-homologous proteins that are found inthe mitochondrial ribosome may serve such a role.The traditional small subunit assembly map,which was determined for E. coli,43may not haveuniversal applicability. S8, one of the central pro-teins in the assembly path, is conserved across3P2O (Table 2), but its RNA binding site is not.Because of the limited knowledge of MRPs, it isnot yet known whether S15 is conserved, althoughit is important for the assembly of the 30 S centraldomain.44The RNA binding site for S15 has beenshown to be crucial for recruitment of the protein,45but this binding site is not conserved at the 3P2Olevel, and it is missing from C. elegans mito-chondria. Perhaps terminal extensions onto theseproteins could fill the gaps left by the loss ofRNA, but it is not yet clear what effects such exten-sions might have on subunit assembly.The remainder of this discussion is devoted toconsidering the contacts between the ribosomeand the tRNAs. When considering 3P2O conserva-tion, this is complicated by the fact that many mito-chondrial tRNAs have truncations in the D andT-stem/loop regions,46so their three-dimensionalstructures are different from conventional tRNAs.Both computer modeling47and transient electric230 Modeling a Minimal Ribosome
  • 17. birefringence experiments48have shown that thealtered secondary structure necessitates substantialconformational changes in the elbow region,leading to a large increase in the angle betweenthe two arms of the tRNA.Examination of contacts between the ribosomalRNA and tRNA (Table 5) shows that these aremost highly conserved at the P-site, and least con-served at the E-site. Contacts with the anticodon,the elbow, and the 30acceptor terminus of theP-site tRNA are all conserved across 3P2O, includ-ing in C. elegans. At the other extreme, conservationat the E-site is weak enough that it is possible that atrue E-site may not exist in all ribosomes, particu-larly those from mitochondria, or, if it exists, itmay have a substantially different structuralorganization.The situation is more complicated at the A-site.Contacts with the anticodon and acceptor terminusare preserved across 3P2O, but contacts betweenthe large subunit and the elbow are not. In the3P2O model, contacts are lost with both theD-loop (residues 17 and 19) and the T-loop(residues 55 and 56), and with the T-stem (residue62). The contact with residue 62 is probably notcritical, since it is not even conserved at the 3Plevel. It is more difficult to interpret the loss of theother four contacts (Table 5); these are conservedacross the three phylogenetic domains, but notacross 3P2O.While the non-standard conformation of mito-chondrial tRNAs might be invoked to explain lossof contacts with the elbow at the A-site, how aresuch contacts then preserved at the P-site? Onepossibility is that the regions of the rRNA respon-sible for these contacts are located in differentpositions in mitochondrial ribosomes than in otherribosomes. Another puzzle is that, since precisetRNA positioning is presumably critical to trans-lational fidelity and efficiency, how can mito-chondrial ribosomes have lost contact with theelbow of the A-site tRNA? Either A-site positioningis not as critical as it is at the P-site, or contact withthe tRNA elbow at the A-site is provided by someother region of the rRNA or by a ribosomalprotein.Materials and MethodsComparative analysisThe sequence alignments used for this analysis aremaintained at the CRW site24†. rRNA sequences weremanually aligned to maximize sequence and structuralidentity using the alignment editor AE2 (T. Macke,Scripps Clinic, San Diego, CA). The rRNA alignmentsare sorted by phylogeny and cell location. The sequencesused in this analysis are at least 90% complete andpublicly available. Specific data for each sequence areaccessible from the CRW site’s relational databasemanagement system (RDBMS).The numbering systems from the E. coli 16 S and 23 SrRNA sequences (GenBank Accession no. J01695) areused as the references for position numbers for both16 S and 23 S rRNAs.Secondary structure and conservation diagrams weredeveloped entirely or in part with the interactivegraphics program XRNA (Weiser & Noller, Universityof California, Santa Cruz). The PostScript files output byXRNA were converted into PDF using ghostscript(version 7.00‡).Computer modeling and graphicsThe models are based on the crystal structures 1FJGfor the 30 S subunit of T. thermophilus,151FFK for the50 S subunit of H. marismortui,14and 1GIX/1GIY for the70 S ribosome from T. thermophilus.17They weregenerated and evaluated using Insight II and QUANTA(Molecular Simulations, Inc.). Areas that are unresolvedin the large subunit crystal structure were modeledusing various techniques. These regions include helix38, helix 69, and the L1 and L11 binding domains of the23 S rRNA. The L11–RNA crystal structure49was super-imposed onto the 50 S structure (1FFK) using tools inthe Insight II program. The L1 binding domain of therRNA was built using the phosphate positions of the70 S structure as a guide. Helices 38 and 69 were builtmanually using Insight. All of these modeled regionswere compared with density maps from cryo-EM studiesof intact 70 S particles.19Helices 38 and 69 of the largesubunit and the L11-binding domain fit the densityrather well while the L1 domain has to be moved slightlyfrom the body of the structure to fit the density.RIBBONS50was used to create the images in Figure 2.It was also used to create image files that were processedby POV-Ray§ to generate Figures 1, 4 and 6.Protein conservationProtein conservation was determined from the websitemaintained by Dr Nikos C. Kyrpides{ at the Universityof Illinois. Information on MRPs is limited to a feworganisms, especially bovine,51yeast52and rat systems.53Therefore, the list of conserved proteins in Table 2 isincomplete and can be expected to grow as more databecome available.For this work, we define protein conservation by thepresence of the protein in the ribosome of the speciesbeing studied. The degree of amino acid or domainconservation within that protein is not considered. It isconserved if homologous proteins are found in thegenomes of divergent species including in the datasetbeing considered for the study (3P or 3P2O).Probability of movementIn evaluating the model of the C. elegans mitochondrialribosome, we have estimated the probability, P(move),that a deletion has forced movement of some region ofthe rRNA to close the resulting gap. This probability is†‡§{ a Minimal Ribosome 231
  • 18. based on distributions of inter-phosphate distancesmeasured in the two crystal structures of the 70 S particle(1GIX and 1GIY). For a deletion where unalignedsequence is available to span the gap, consecutivephosphate distances are measured (p þ n, where n is thesequential phosphate number determined by the lengthof the unaligned sequence) generating a distribution ofdistances. These can be compared with distances of con-secutive phosphate groups created by this deletion, anda probability of movement is then calculated.Two examples are shown in Figure 5. The first showsthe distribution of distances found in the crystal struc-ture for phosphate groups that are separated by eightnucleotides, corresponding to a connection betweenphosphate i and phosphate i þ 9. Within the secondarystructure of C. elegans the absence of helix 94 of the largesubunit leaves eight nucleotides of unaligned sequenceto span a gap of 43 A˚ . The distribution of distancesfound in the crystal structure shows that for eightnucleotides, 99.3% of the population has a distance lessthan 43 A˚ . In this case, then, P(move) would be 0.993.The second example in Figure 5 shows the distributionof distances for phosphate groups that are spaced by 24nucleotides, connecting phosphate i with phosphatei þ 25. In C. elegans, the deletion of helices 81–88 (resi-dues 2259–2421) results in a gap of 26 A˚ to be spannedby 24 nucleotides of unaligned sequence. Only 19.5% ofthe distances in the population are smaller than this, soP(move) would only be 0.195. In this case the nucleotidesat the ends of the gap could be connected without diffi-culty, and the affected domains could easily have thesame relative positions in C. elegans as in H. marismortui.Supplemental informationFigures 1 and 4, showing phylogenetic conservationand the alignment of C. elegans mitochondrial rRNA,can be viewed from several perspectives in movies atthe Harvey laboratory website ( Secondary structures andadditional information about the comparative analysescan be found at the CRW site maintained by the Gutelllaboratory ( Conservation of tRNA binding siteswere studied using a distance criterion (8.5 and 10.0 A˚ )to determine the conservation of residues near themRNA and tRNAs in the crystal structures (1GIX and1GIY). The findings are comparable to the results pre-sented in Table 5.AcknowledgmentsWe acknowledge Jung C. Lee for work with tRNA con-tacts and Dr Joachim Frank for helpful discussions. Thiswork was funded by grants from the National Instituteof Health to S.C.H. (GM-53827), R.R.G. (GM 48207), andR.K.A. (GM 61576).References1. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N.& Altman, S. (1983). The RNA moiety of ribonucleaseP is the catalytic subunit of the enzyme. Cell, 35,849–857.2. Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J.,Gottschling, D. E. & Cech, T. R. (1982). Self-splicingRNA: autoexcision and autocyclization of the riboso-mal RNA intervening sequence of Tetrahymena. Cell,31, 147–157.3. Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz,T. A. (2000). The structural basis of ribosome activityin peptide bond synthesis. Science, 289, 920–930.4. Carter, A. P., Clemons, W. M., Brodersen, D. E., Mor-gan-Warren, R. J., Wimberly, B. T. & Ramakrishnan,V. (2000). Functional insights from the structure ofthe 30S ribosomal subunit and its interactions withantibiotics. Nature, 407, 340–348.5. Fourmy, D., Recht, M. I., Blanchard, S. C. & Puglisi, J.D. (1996). Structure of the A site of Escherichia coli 16Sribosomal RNA complexed with an aminoglycosideantibiotic. Science, 274, 1367–1371.6. VanLoock, M. S., Easterwood, T. R. & Harvey, S. C.(1999). Major groove binding of the tRNA/mRNAcomplex to the 16 S ribosomal RNA decoding site.J. Mol. Biol. 285, 2069–2078.7. Agrawal, R. K., Heagle, A. B., Penczek, P., Grassucci,R. A. & Frank, J. (1999). EF-G-dependent GTPhydrolysis induces translocation accompanied bylarge conformational changes in the 70S ribosome.Nature Struct. Biol. 6, 643–647.8. Frank, J. & Agrawal, R. K. (2000). A ratchet-like inter-subunit reorganization of the ribosome during trans-location. Nature, 406, 318–322.9. Agrawal, R. K., Penczek, P., Grassucci, R. A. & Frank,J. (1998). Visualization of elongation factor G on theEscherichia coli 70S ribosome: the mechanism oftranslocation. Proc. Natl Acad. Sci. USA, 95,6134–6138.10. Agrawal, R. K., Spahn, C. M., Penczek, P., Grassucci,R. A., Nierhaus, K. H. & Frank, J. (2000). Visualiza-tion of tRNA movements on the Escherichia coli 70Sribosome during the elongation cycle. J. Cell Biol.150, 447–460.11. Stark, H., Rodnina, M. V., Rinke-Appel, J.,Brimacombe, R., Wintermeyer, W. & van Heel, M.(1997). Visualization of elongation factor Tu on theEscherichia coli ribosome. Nature, 389, 403–406.12. Ogle, J. M., Brodersen, D. E., Clemons, W. M., Jr,Tarry, M. J., Carter, A. P. & Ramakrishnan, V. (2001).Recognition of cognate transfer RNA by the 30S ribo-somal subunit. Science, 292, 897–902.13. Lodmell, J. S. & Dahlberg, A. E. (1997). A confor-mational switch in Escherichia coli 16S ribosomalRNA during decoding of messenger RNA. Science,277, 1262–1267.14. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz,T. A. (2000). The complete atomic structure of thelarge ribosomal subunit at 2.4 A˚ resolution. Science,289, 905–920.15. Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr,Morgan-Warren, R. J., Carter, A. P., Vonrhein, C. et al.(2000). Structure of the 30S ribosomal subunit.Nature, 407, 327–339.16. Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J.,Gluehmann, M., Janell, D. et al. (2000). Structure offunctionally activated small ribosomal subunit at3.3 A˚ resolution. Cell, 102, 615–623.17. Yusupov, M. M., Yusupova, G. Z., Baucom, A.,Lieberman, K., Earnest, T. N., Cate, J. H. & Noller,H. F. (2001). Crystal structure of the ribosome at5.5 A˚ resolution. Science, 292, 883–896.232 Modeling a Minimal Ribosome
  • 19. 18. Agrawal, R. K. & Frank, J. (1999). Structural studiesof the translational apparatus. Curr. Opin. Struct.Biol. 9, 215–221.19. Gabashvili, I. S., Agrawal, R. K., Spahn, C. M.,Grassucci, R. A., Svergun, D. I., Frank, J. & Penczek,P. (2000). Solution structure of the E. coli 70S ribo-some at 11.5 A˚ resolution. Cell, 100, 537–549.20. Ramakrishnan, V. & Moore, P. B. (2001). Atomicstructures at last: the ribosome in 2000. Curr. Opin.Struct. Biol. 11, 144–154.21. Gutell, R. R., Larsen, N. & Woese, C. R. (1994).Lessons from an evolving rRNA: 16S and 23S rRNAstructures from a comparative perspective. Microbiol.Rev. 58, 10–26.22. Gutell, R. R., Weiser, B., Woese, C. R. & Noller, H. F.(1985). Comparative anatomy of 16S-like ribosomalRNA. Prog. Nucl. Acid. Res. Mol. Biol. 32, 155–216.23. Gutell, R. R. (1992). Evolutionary characteristics of16S and 23S rRNA structures. In Proceedings of theConference on the Origin and Evolution of Prokaryoticand Eukaryotic Cells. The Origin and Evolution of theCell (Hartman, H. & Matsuno, K., eds), WorldScientific, Singapore.24. Cannone, J. J., Subramanian, S., Schnare, M. N.,Collett, J. R., D’Souza, L. M., Du, Y. et al. (2002). TheComparative RNA Web (CRW) site: an online data-base of comparative sequence and structure infor-mation for ribosomal, intron, and other RNAs. BMCBioinformatics, 3, 2.25. Frank, J., Zhu, J., Penczek, P., Li, Y., Srivastava, S.,Verschoor, A. et al. (1995). A model of protein syn-thesis based on cryo-electron microscopy of theE. coli ribosome. Nature, 376, 441–444.26. Malhotra, A., Penczek, P., Agrawal, R. K., Gabashvili,I. S., Grassucci, R. A., Junemann, R. et al. (1998).Escherichia coli 70 S ribosome at 15 A˚ resolution bycryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein. J. Mol. Biol. 280,103–116.27. Wuyts, J., Van de Peer, Y. & De Wachter, R. (2001).Distribution of substitution rates and location ofinsertion sites in the tertiary structure of ribosomalRNA. Nucl. Acids Res. 29, 5017–5028.28. Yamaguchi, K. & Subramanian, A. R. (2000). Theplastid ribosomal proteins. Identification of all theproteins in the 50 S subunit of an organelle ribosome(chloroplast). J. Biol. Chem. 275, 28466–28482.29. Yamaguchi, K., von Knoblauch, K. & Subramanian,A. R. (2000). The plastid ribosomal proteins. Identifi-cation of all the proteins in the 30S subunit of anorganelle ribosome (chloroplast). J. Biol. Chem. 275,28455–28465.30. Agrawal, R. K., Linde, J., Sengupta, J., Nierhaus, K.H. & Frank, J. (2001). Localization of L11 protein onthe ribosome and elucidation of its involvement inEF-G-dependent translocation. J. Mol. Biol. 311,777–787.31. Koc, E. C., Burkhart, W., Blackburn, K., Koc, H.,Moseley, A. & Spremulli, L. L. (2001). Identificationof four proteins from the small subunit of the mam-malian mitochondrial ribosome using a proteomicsapproach. Protein Sci. 10, 471–481.32. Koc, E. C., Burkhart, W., Blackburn, K., Moyer, M. B.,Schlatzer, D. M., Moseley, A. & Spremulli, L. L.(2001). The large subunit of the mammalian mito-chondrial ribosome. Analysis of the complement ofribosomal proteins present. J. Biol. Chem. 276,43958–43969.33. Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada,T., Ueda, T., Wada, A. & Watanabe, K. (2001).Structural compensation for the deficit of rRNAwith proteins in the mammalian mitochondrial ribo-some. J. Biol. Chem. 276, 21724–21736.34. Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada,T., Ueda, T., Wada, A. & Watanabe, K. (2001). Struc-tural compensation for the deficit of rRNA with pro-teins in the mammalian mitochondrial ribosome.Systematic analysis of protein components of thelarge ribosomal subunit from mammalian mito-chondria. J. Biol. Chem. 276, 21724–21736.35. Okimoto, R., MacFarlane, J. L., Clary, D. O. &Wolstenholme, D. R. (1992). The mitochondrial gen-omes of two nematodes, Caenorhabditis elegans andAscaris suum. Genetics, 130, 471–498.36. Morosyuk, S. V., Cunningham, P. R. & SantaLucia, J.(2001). Structure and function of the conserved 690hairpin in Escherichia coli 16 S ribosomal RNA. II.J. Mol. Biol. 307, 197–211.37. Morosyuk, S. V., SantaLucia, J. & Cunningham, P. R.(2001). Structure and function of the conserved 690hairpin in Escherichia coli 16 S ribosomal RNA. III.J. Mol. Biol. 307, 213–228.38. Gabashvili, I. S., Agrawal, R. K., Grassucci, R.,Squires, C. L., Dahlberg, A. E. & Frank, J. (1999).Major rearrangements in the 70S ribosomal 3Dstructure caused by a conformational switch in 16Sribosomal RNA. EMBO J. 18, 6501–6507.39. VanLoock, M. S., Agrawal, R. K., Gabashvili, I. S., Qi,L., Frank, J. & Harvey, S. C. (2000). Movement of thedecoding region of the 16 S ribosomal RNA accom-panies tRNA translocation. J. Mol. Biol. 304, 507–515.40. Velichutina, I. V., Dresios, J., Hong, J. Y., Li, C.,Mankin, A., Synetos, D. & Liebman, S. W. (2000).Mutations in helix 27 of the yeast Saccharomycescerevisiae 18S rRNA affect the function of the decod-ing center of the ribosome. RNA, 6, 1174–1184.41. Lee, K., Varma, S., SantaLucia, J., Jr & Cunningham,P. R. (1997). In vivo determination of RNA struc-ture–function relationships: analysis of the 790 loopin ribosomal RNA. J. Mol. Biol. 269, 732–743.42. Yusupova, G. Z., Yusupov, M. M., Cate, J. H. &Noller, H. F. (2001). The path of messenger RNAthrough the ribosome. Cell, 106, 233–241.43. Held, W. A., Ballou, B., Mizushima, S. & Nomura, M.(1974). Assembly mapping of 30S ribosomal proteinsfrom Escherichia coli. Further studies. J. Biol. Chem.249, 3103–3111.44. Agalarov, S. C., Sridhar Prasad, G., Funke, P. M.,Stout, C. D. & Williamson, J. R. (2000). Structure ofthe S15,S6,S18–rRNA complex: assembly of the 30Sribosome central domain. Science, 288, 107–113.45. Serganov, A., Benard, L., Portier, C., Ennifar, E.,Garber, M., Ehresmann, B. & Ehresmann, C. (2001).Role of conserved nucleotides in building the 16 SrRNA binding site for ribosomal protein S15. J. Mol.Biol. 305, 785–803.46. Wolstenholme, D. R., MacFarlane, J. L., Okimoto, R.,Clary, D. O. & Wahleithner, J. A. (1987). BizarretRNAs inferred from DNA sequences of mito-chondrial genomes of nematode worms. Proc. NatlAcad. Sci. USA, 84, 1324–1328.47. Steinberg, S. & Cedergren, R. (1994). Structural com-pensation in atypical mitochondrial tRNAs. NatureStruct. Biol. 1, 507–510.48. Frazer-Abel, A. A. & Hagerman, P. J. (1999). Determi-nation of the angle between the acceptor andModeling a Minimal Ribosome 233
  • 20. anticodon stems of a truncated mitochondrial tRNA.J. Mol. Biol. 285, 581–593.49. Wimberly, B. T., Guymon, R., McCutcheon, J. P.,White, S. W. & Ramakrishnan, V. (1999). A detailedview of a ribosomal active site: the structure of theL11–RNA complex. Cell, 97, 491–502.50. Carson, M. (1997). Ribbons. Methods Enzymol. 277,493–505.51. Pietromonaco, S. F., Denslow, N. D. & O’Brien, T. W.(1991). Proteins of mammalian mitochondrial ribo-somes. Biochimie, 73, 827–835.52. Graack, H. R. & Wittmann-Liebold, B. (1998).Mitochondrial ribosomal proteins (MRPs) of yeast.Biochem. J. 329, 433–448.53. Goldschmidt-Reisin, S., Kitakawa, M., Herfurth, E.,Wittmann-Liebold, B., Grohmann, L. & Graack, H.R. (1998). Mammalian mitochondrial ribosomalproteins. N-terminal amino acid sequencing, charac-terization, and identification of corresponding genesequences. J. Biol. Chem. 273, 34828–34836.Edited by D. E. Draper(Received 29 April 2002; received in revised form 4 June 2002; accepted 5 June 2002)234 Modeling a Minimal Ribosome