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A Structural Model for the Large Subunit of the Mammalian Mitochondrial Ribosome Jason A. Mears 1,2 , Manjuli R. Sharma 3 , Robin R. Gutell 4 Amanda S. McCook 1 , Paul E. Richardson 5 , Thomas R. Caulfield 6 Rajendra K. Agrawal 3,7 and Stephen C. Harvey 1 * 1 Department of Biology, Georgia Institute of Technology, Atlanta GA 30332, USA 2 Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham, Birmingham AL 35295, USA 3 Division of Molecular Medicine, Wadsworth Center New York State Department of Health, Albany, NY 12201 USA 4 Institute for Cellular and Molecular Biology and Section of Integrative Biology University of Texas at Austin 2500 Speedway, Austin, TX 78712, USA 5 Department of Chemistry Coastal Carolina University Conway, SC 29528, USA 6 Department of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA 7 Department of Biomedical Sciences, State University of New York at Albany, Albany NY 12201, USA Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1 A ˚ resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures. q 2006 Elsevier Ltd. All rights reserved. Keywords: ribosome; mitochondrial evolution; rRNA; comparative sequence analysis; molecular fitting *Corresponding author 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. J. A. M. and M.R.S. contributed equally to this work. Abbreviations used: mitoribosome, mitochondrial ribosome; mito-rRNA, mitochondrial ribosomal RNA; MRPs, mitochondrial ribosomal proteins; LSU, large subunit; PTC, peptidyl transferase center; cryo-EM, cryo-electron microscopy; SRL, sarcin-ricin loop; ASF, the A-site finger motif; 5S-BD, the 5 S rRNA-binding domain; L1-BD, the L1-binding domain. E-mail address of the corresponding author: [email protected] doi:10.1016/j.jmb.2006.01.094 J. Mol. Biol. (2006) 358, 193–212

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Mears J.A., Sharma M.R., Gutell R.R., McCook A.S., Richardson P.E., Caulfield T.R., Agrawal R.K., and Harvey S.C. (2006). A Structural Model for the Large Subunit of the Mammalian Mitochondrial Ribosome. Journal of Molecular Biology, 358(1):193-212.


  • 1. A Structural Model for the Large Subunit ofthe Mammalian Mitochondrial RibosomeJason A. Mears1,2, Manjuli R. Sharma3, Robin R. Gutell4Amanda S. McCook1, Paul E. Richardson5, Thomas R. Cauleld6Rajendra K. Agrawal3,7and Stephen C. Harvey1*1Department of Biology, GeorgiaInstitute of Technology, AtlantaGA 30332, USA2Department of Biochemistryand Molecular GeneticsUniversity of Alabama atBirmingham, BirminghamAL 35295, USA3Division of MolecularMedicine, Wadsworth CenterNew York State Department ofHealth, Albany, NY 12201USA4Institute for Cellular andMolecular Biology and Sectionof Integrative BiologyUniversity of Texas at Austin2500 Speedway, Austin, TX78712, USA5Department of ChemistryCoastal Carolina UniversityConway, SC 29528, USA6Department of Chemistry &Biochemistry, Georgia Instituteof Technology, Atlanta, GA30332, USA7Department of BiomedicalSciences, State University ofNew York at Albany, AlbanyNY 12201, USAProtein translation is essential for all forms of life and is conducted by amacromolecular complex, the ribosome. Evolutionary changes in proteinand RNA sequences can affect the 3D organization of structural features inribosomes in different species. The most dramatic changes occur in animalmitochondria, whose genomes have been reduced and altered signicantly.The RNA component of the mitochondrial ribosome (mitoribosome) isreduced in size, with a compensatory increase in protein content. Untilrecently, it was unclear how these changes affect the 3D structure of themitoribosome. Here, we present a structural model of the large subunit ofthe mammalian mitoribosome developed by combining molecularmodeling techniques with cryo-electron microscopic data at 12.1 Aresolution. The model contains 93% of the mitochondrial rRNA sequenceand 16 mitochondrial ribosomal proteins in the large subunit of themitoribosome. Despite the smaller mitochondrial rRNA, the spatialpositions of RNA domains known to be involved directly in proteinsynthesis are essentially the same as in bacterial and archaeal ribosomes.However, the dramatic reduction in rRNA content necessitates evolution ofunique structural features to maintain connectivity between RNA domains.The smaller rRNA sequence also limits the likelihood of tRNA bindingat the E-site of the mitoribosome, and correlates with the reduced size ofD-loops and T-loops in some animal mitochondrial tRNAs, suggestingco-evolution of mitochondrial rRNA and tRNA structures.q 2006 Elsevier Ltd. All rights reserved.Keywords: ribosome; mitochondrial evolution; rRNA; comparativesequence analysis; molecular tting*Corresponding author0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. J. A. M. and M.R.S. contributed equally to this work.Abbreviations used: mitoribosome, mitochondrial ribosome; mito-rRNA, mitochondrial ribosomal RNA; MRPs,mitochondrial ribosomal proteins; LSU, large subunit; PTC, peptidyl transferase center; cryo-EM, cryo-electronmicroscopy; SRL, sarcin-ricin loop; ASF, the A-site nger motif; 5S-BD, the 5 S rRNA-binding domain; L1-BD, theL1-binding domain.E-mail address of the corresponding author: [email protected]:10.1016/j.jmb.2006.01.094 J. Mol. Biol. (2006) 358, 193212

2. IntroductionThe mammalian mitochondrial ribosome (mitor-ibosome) is responsible for synthesis of 13 mito-chondrial gene products that are essentialcomponents of the complexes involved in oxidativephosphorylation.1,2The importance of these pro-teins in generating ATP places a signicant burdenof accuracy on the mitoribosome. The mitochon-drion also plays a crucial role in the initiation ofapoptosis,3and mitochondrial defects have beenimplicated in a wide variety of degenerativediseases, aging, and cancer.4However, evolutionarypressures to maintain nuclear control of cellularmetabolism following endosymbiosis5may beresponsible for the signicant reduction in animalmitochondrial ribosomal RNA (mito-rRNA)sequence when compared to bacteria, archaea andeukaryotes. This reduction is compensated by anincrease in the size and number of mitochondrialribosomal proteins (MRPs),68whose genes areunder nuclear control.9Even though mitochondria are believed to havedescended from an endosymbiotic eubacter-ium,10,11the structural components of theirribosomes are noticeably different. The ratio ofprotein to rRNA mass in animal mitochondria (2:1)is inverted from the ratio found in bacterial andarchaeal ribosomes (1:2). A decrease in particledensity is therefore observed in sedimentationexperiments, yielding a 55 S value for the intactbovine mitoribosome compared to 70 S in bacteriaand archaea. The decreased sedimentation coef-cient can also be attributed to a more porousstructure in mitochondrial ribosomes.12,13The bovine 55 S mitoribosome is comprised oftwo asymmetric subunits, a small (28 S) subunitand large (39 S) subunit. The small subunit containsa 12 S rRNA (955 nucleotides) with 29 pro-teins,6,14,15and the large subunit contains a 16 SrRNA (1571 nucleotides) with 48 proteins.68All ofthe MRPs are encoded by rapidly evolving nucleargenes,16while the mitochondrial rRNAs, which arealso evolving at a rapid rate,5are encoded by themitochondrial genome and are transcribed withinthe mitochondrion. For comparison, the bacterialand archaeal ribosomes are also comprised of twoasymmetric subunits, but the small (30 S) subunitcontains a 16 S rRNA (1500 nucleotides, on average)with roughly 20 proteins and the large (50 S)subunit contains two ribosomal RNA (rRNA)components, 5 S rRNA (120 nucleotides, on aver-age) and 23 S rRNA (3000 nucleotides, on average),with more than 30 proteins. tRNAs migrate throughthree relatively stable binding sites in cytoplasmicribosomes during translation: the A-site (ami-noacyl), the P-site (peptidyl), and the E-site (exit).Here, we focus our attention on the structure ofthe large subunit of the mammalian mitoribosome.The large subunit (LSU) rRNA is responsible forcatalysis of peptide bond formation. Recent studiessuggest that the mechanism of peptide bondformation can be attributed to positioning transferRNA (tRNA) substrates charged with amino acidresidues in a specied proximity during thereaction.17,18The 20-OH of residue A76 of theP-site tRNA has been proposed as the catalyticcomponent.19The LSU rRNA domain V, whichcontains the peptidyl transferase center (PTC), islargely conserved through all organisms.20Many ofthe rRNA regions of domain IV that are involved intRNA and inter-subunit interactions21are alsopreserved.20The ribosomal protein L11-binding domain(L11-BD) within the LSU rRNA domain II and thesarcin-ricin loop within domain VI, which togetherconstitute the GTPase-associated center of theribosome that interacts with translation cofactors(EF-Tu, EF-G, RF3, etc.),22,23are also conserved.However, the sequences and structures that connectthe central core and these peripheral structuralelements are truncated in several mitochondrialLSU rRNAs.24On the other side of the LSU,dynamic motions of the L1 protein and associatedrRNA have been proposed to affect E-site occu-pancy on the ribosome directly.2527Together,these functional domains work in concert duringprokaryotic translation, and structural studies arebeginning to elucidate the mechanisms required forprotein synthesis. Even so, it is unclear how theevolving mitoribosome compensates for the largereduction in rRNA sequence while maintaining theprecision that protein synthesis demands.A recent cryo-electron microscopy (cryo-EM)study has provided the rst detailed structure ofthe mammalian mitoribosome.13Previously deter-mined X-ray structures21,25,28of large ribosomalsubunits were compared with the mitoribosomestructure, but no effort was made to optimize the tof the structures to the EM density. Here, a higherresolution (12.1 A ) provides an improved rRNAprotein separation in the EM density, allowing adetailed tting of modeled structures. As docu-mented previously,13the structure has severalunique features when compared with structures ofcytoplasmic ribosomes from prokaryotic andeukaryotic organisms. Also, the additional proteincontent does not compensate for the missing RNAsequence, as has been proposed.7,16Instead, manyof the additional proteins assume unique positions inthe mitoribosome structure, thereby leaving regionsof structure vacant where rRNA helices present inbacteria have been deleted in the mitoribosome. Also,in contrast to the characteristics of cytoplasmicribosomes, no tRNA was found at the putative exitsite (E-site) of the 55 S mitoribosome, suggesting thatthe E-site is either very weak or non-existent in themitoribosome, consistent with suggestions from acomparative analysis of mitochondrial and nuclear-encoded ribosomes.24In this study, we analyze the structural pertur-bations in the regions of the mammalian mitoribo-some that have signicant changes in size andsequence of rRNA and proteins. We utilize a varietyof methods, including bioinformatics, to study theevolution of RNA and protein sequences, structural194 Structural Evolution in Mitochondrial Ribosomes 3. biology (cryo-EM), and novel computational toolsto unify the results in a detailed 3D model of themitoribosome. In most cases, the reduction in mito-rRNA sequence does not alter the 3D spatiallocation of conserved rRNA helical elements.Thus, many of the interactions between themitoribosome, tRNAs and protein cofactors duringthe translation cycle are preserved. However, someof the reductions in rRNA sequence result in theloss or reorientation of functional domains, indicat-ing structures in the bacterial and archaeal ribo-somes that are highly variable and may not beessential for translation. We have modeled thestructures and placement for 16 MRPs that havesufcient levels of sequence identity with homolo-gous prokaryotic proteins whose structures havebeen determined by X-ray crystallography. Thiswork provides a rst step towards assigningstructure to the protein mass of the mitochondrialribonucleoprotein structure. Additional structuraland biochemical studies are required to predict theplacement and conformations of the remaining,unmodeled proteins, especially those that areunique to the mitoribosome.ResultsHomology modeling of mitochondrial rRNA(mito-rRNA)Based on the assumption that the secondary andtertiary structures for rRNA molecules are mostlyconserved for all organisms, we have usedcomparative sequence analysis to identifysequence conservation and variation, and deter-mine similar structural elements that are present insets of distantly related and closely related rRNAsequences. Previous success in determining thesecondary structures for the small and largesubunit rRNAs29lends condence to the secondarystructure model for the mitochondrial LSU rRNAderived from comparative sequence analysis(Figure 1). Approximately 86% of the Bos taurusmito-rRNA can be assigned to structural featurespresent in the archaeal Haloarcula marismortuisecondary structure, available from the Compara-tive RNA Website.20The B. taurus mito-rRNA(Figure 1(a)) has many features in common withall nuclear-encoded rRNAs, including the peptidyltransfer center (PTC), the sarcin-ricin loop (SRL),and L11-BD. However, many differences areevident, because the size of the LSU mito-rRNAis reduced by almost half compared to bacteria.Many of the helical structures in domains I and IIIare lost, as are the A-site nger motif (ASF) indomain II, and the L1- and the 5 S rRNA-bindingdomains (L1-BD and 5S-BD, respectively) indomain V.Starting with the secondary structure for the largesubunit rRNA of the bovine mitoribosome(Figure 1(a)), we have used homology modeling30to generate a structural model for the mito-rRNA ofthe LSU of the mitoribosome. The X-ray crystallo-graphic structures of large ribosomal subunits fromH. marismortui,28an archaeon, and Deinococcusradiodurans,25a bacterium, provide homologousrRNA structures to guide the modeling. For ourstudies, we primarily used the H. marismortuistructure (PDB accession code 1JJ2) as the templatefor modeling the mitochondrial rRNA structure.Helices and loop structures that are conserved inmitochondria are modeled on the basis of geometrydetermined by X-ray crystallography for thearcheon (see Methods). Three signicant differenceswere explored: (1) identical secondary structureelements composed of the same base-pairings andunpaired nucleotides are modeled one for one,when the sequences are identical or vary incomposition. The nucleotides from H. marismortuiare replaced by corresponding nucleotides in themito-rRNA. These changes do not affect thebackbone or sugar orientation of the bases. (2) Forcanonical base-pairs that are replaced by non-canonical pairs (i.e. G$U or G$A), the latter pair issuperposed on the canonical pair. The same methodis used for non-canonical pairs that are replaced bycanonical pairs. None of these changes in base-pairtypes distort the helical geometry severely, anddifferences in the backbone interactions withneighboring nucleotides are easily resolved with around of energy minimization. (3) For moredramatic differences between the two secondarystructures, including changes in the size of loopsand bulges, the mitochondrial sequence is modeledfrom previously determined X-ray crystallographystructures of RNA with similar sequence (availablefrom the RCSB Protein Data Bank31).Structure renement and validationThe homology model of the mito-rRNA providesa starting structure with helical positions based onsimilarities to prokaryotic ribosomes as determinedby comparative sequence analysis. Informationfrom a cryo-EM study13provides additional,experimental restraints that can be incorporatedusing YAMMP,32our in-house molecular modelingpackage. The software includes a rigid-body MonteCarlo module with simulated annealing that we useto rene our model. The vector lattice (VLAT)component of YAMMP generates a forceeld termthat denes the electron density from experimentalstudies as a 3D potential, providing a score for thet of the model to the density.Initially, the complete model for the mitochon-drial rRNA is treated as a single rigid unit using areduced representation.33The starting model con-tains pseudoatoms representing the phosphatepositions of each nucleotide in the RNA homologymodel (P-atoms). To get an initial placement of themodel in the EM density, the rigid unit wassubjected to Monte Carlo renement with simu-lated annealing (see Methods). A good t isobtained, suggesting that the structural organiza-tion is largely conserved in the mitoribosome, withStructural Evolution in Mitochondrial Ribosomes 195 4. Figure 1. rRNA secondary structure comparison. (a) Bos taurus mitochondrial rRNA secondary structure based oncomparative sequence analysis. (b) Secondary structure of H. marismortui 23 S rRNA. Regions that align with themitochondrial rRNA are highlighted in red, and those that are absent from the mitoribosome are shown in black.Some relevant functional regions are labeled (green), and six domains of the 23 S rRNA are identied with romannumerals.196 Structural Evolution in Mitochondrial Ribosomes 5. small changes due to differences in sequence andsize of some helices and connecting regions.The mito-rRNA model was then divided into 52rigid units, based on conserved helical structures(Figure 2), and the structure was rened to thecomplete large subunit density with multiple rigid-body Monte Carlo with simulated annealing. Eachrigid unit is topologically connected to neighboringunits using one of two bond-types. (1) Strong bondsare used to connect adjacent conserved structuresthat are separated by distances corresponding to thenormal phosphatephosphate distances betweenneighboring bases (w67 A ). (2) Weak bondsconnect nucleotides separated by a gap in themodel due to a lack of secondary structureinformation (Figure 2, grey regions). The weakerbonds allow greater freedom for the connectedstructures, while maintaining reasonable connec-tivity during the Monte Carlo renement. Non-bond interactions with 7.5 A exclusion diameterswere used for every P-atom in the structure toprevent structural overlap.All-atom models for the 52 units were thensuperposed on the nal reduced representationmodels t to the EM density, given that the reducedrRNA units were treated as rigid bodies andmaintained their geometries during renement.The all-atom models for each unit were covalentlylinked, except for the regions where gaps occur(Figure 2, broken grey lines); and a round of energyminimization was used to resolve structural dis-crepancies caused by rearrangements during rigid-body renement.The t of this model was evaluated using theseparated rRNA density. With few exceptions (seebelow), the modeled structures t the density well.This is due to the large conservation of structuralelements found in both mitochondrial and prokar-yotic rRNAs (seen in Figure 1). H27 in domain II(Figure 2) does have a slightly different positionrelative to the rest of the rRNA structure because ofthe increased size in the adjacent loop whencompared to archaea. This does not affect inter-actions with other conserved rRNA regions. Also,H75 (Figure 2) has a change in sequence that placesan unpaired nucleotide on the opposing side of thehelix when compared with archaea. This changeforces the helix into a position that places the L1protein away from the E-site (discussed below). Themost dramatic changes in rRNA structure are dueto sequences that are missing or unique regionswhose structures could not be determined bycomparative sequence analysis.Additional and alternative RNA secondarystructure predictionsThe only helices in the LSU rRNA comparativemodel that do not t the EM density afterrenement are in domains I and III (in Figure 2,helix 13, the end of helix 50 and helix 59.1 didnot nd a reasonable t to the cryo-EM density).We therefore explored the possibility that these tworegions of the rRNA have alternative folds usingAlifold.34In contrast to comparative analysis of therRNAs that identies base-pairs from patterns ofvariation and covariation in a set of alignedsequences,35the Alifold34program combines ther-modynamic and comparative analyses to predictRNA secondary structure. A multiple sequencealignment was created for a set of mammalianmitochondrial LSU rRNAs, that includes B. taurusand related organisms (see Methods). The Alifoldprogram identied helices that are thermodynami-cally stable and present in the set of alignedsequences. This suggests a secondary structure inthe region of helices H50 and H59.1 (Figure 3, greennucleotides) that differs somewhat from thatproposed by comparative analysis (Figure 2). Themodeled structure matches closely the RNA separ-ated EM density (Figure 4(c)). This suggests one oftwo possibilities: (1) that after endosymbiosis thesequence in domain III of the bovine mito-rRNAhas evolved to generate a somewhat different 3Dstructure; or (2) the structure determined by cryo-EM represents a conformational change in thisregion from the structure predicted by comparativesequence analysis. We cannot rule out eitherpossibility, but our model is based on the alternativesecondary structure (shown in Figure 3) that bestts the cryo-EM density. We examined alternativesecondary structures for the unstructured regions ofdomain I (including helix 13), but we were unable tond secondary structure predictions that could beplaced in the EM density with condence.Part of the region in domain II that connects theL11-BD to the highly conserved core of the LSU hasfewer nucleotides in the mammalian mitochondriathan the corresponding region in all nuclear-encoded LSU rRNAs (Figure 1). No base-pairingor helices that are shared in the mammalianmitochondria LSU rRNA were identied withcomparative and covariation analysis for thistruncated region. To examine the possibility ofadditional secondary structure in this region, weused the mfold36thermodynamic folding programto predict helices to expand the model fromcomparative analysis. Several thermodynamicallystable secondary structure helices were identied,and we tested each of these by examining their tsto the RNA separated cryo-EM density. From this,we were able to generate a 3D model that matchesthe cryo-EM density very well in this region(Figure 4(a)). This model reveals how, in spite ofthe drastic reduction in rRNA size, the L11-BD ofthe mito-rRNA can remain on the periphery of themitoribosome. A previous modeling study24pro-posed a movement of L11-BD closer to the core ofthe LSU during the course of evolution in theCaenorhabditis elegans mitoribosome, because nopreviously characterized RNA structure of similarsequence and length was known to span such alarge distance. No such movement is necessary inthe mammalian mitochondria for two reasons: (1)the sequence is not as reduced in mammalianmitochondria as in C. elegans; and (2) the uniqueStructural Evolution in Mitochondrial Ribosomes 197 6. Figure 2. Rigid-body renement of the rRNA model. A total of 52 independent rigid units were dened for renement of the RNA structure using Monte Carlo withsimulated annealing (see Methods). Regions are colored to represent the distinct units that were used for the renement. Unmodeled regions are represented as broken greylines. Most modeled helices maintain a similar structure and relative location when compared with their bacterial homologs. Helices 27 and 75 are highlighted because ofaltered conformations due to changes in sequence. Helices 13, 50 and 59.1, proposed by comparative sequence analysis (, did not t the EM density.We propose an alternative structure in the region with helices 50 and 59.1 in domain III (see Figures 3 and 4). 7. Figure 3. Proposed secondary structure for the mammalian mitochondrial LSU rRNA. The structure is presented as modeled with regions predicted by comparativesequence analysis (blue) comprising roughly 86% of the secondary structure. Additional secondary structure was predicted using mfold34(orange) to extend the structure forregions where comparative analysis does not predict secondary structure. Alifold34was used to predict an alternative secondary structure in domain III (green) that differsslightly from that predicted by comparative analysis, but matches the cryo-EM density more closely.13 8. structure found in the mammalian mitoribosomeextends from the core of the subunit to theperiphery (w80 A ) by restricting base-pairing andallowing the RNA strand to stretch without theconstraints of helical geometry. In fact, a largeportion of the sequence near the L11-BD does notform helical base-pairs, because the sequenceconsists almost entirely of adenine and cytosinebases (one uracil and no guanine, Figure 3(a)).Cryo-EM density connecting the L11-BD with therest of the rRNA is relatively thin, which suggestsconformational variability in the region, consistentwith non-canonical interactions.A similar method was used to predict thesecondary structure of an rRNA helix in domainV, which extends to the L1-binding domain (L1-BD).This region of the LSU rRNA is truncated in themammalian mitochondrion, and no base-pairing ispredicted at its apex in the mammalian mitochon-drial comparative structure model (Figure 1(a)).This truncation implies that L1 does not bind to thesame RNA site, despite the conservation of L1 inmammalian mitoribosomes.37Thermodynamic pre-dictions indicate that a single stem-loop structure isfeasible (Figure 3(b)), and modeling places the RNAin close proximity with the L1 protein (Figure 4(b)).For this reason, an RNA:protein interaction isprobably maintained, with the large bulge in thehairpin easily tting the cleft of the L1 protein.38We attempted to determine energetically stablehelices in the remaining regions of the B. taurus LSUrRNA that did not have helices in the comparativestructure model (Figure 3, grey lettering). Unfortu-nately, no common structures were found. In fact,no base-pairings were predicted by mfold36for thelarge loops in domains II and III, because thesesequences lack the nucleic acid base diversityrequired for canonical pairing (no guanine andfew uracil bases). The abundance of adenine andcytosine bases in these unstructured regionssuggests that these regions do not contain regularbase-pairing and helices. The same is true for thesingle-stranded region in domain VI (Figure 3, oneuracil and no guanine). The unmodeled regions indomain I are not completely devoid of guanine, butthey are also very G-poor. Neither Alifold nor mfoldwas able to predict common secondary structuresfor this sequence. We have therefore elected not tomodel these regions. Their structures are probablyunique to mitoribosomes.The nal model of mito-rRNAIn total, we have modeled 93% of the mito-rRNAsequence in three dimensions (Figure 5). Much ofthe 16 S mito-rRNA is conserved in domain IV(Figure 5, green), which lies at the interface betweenFigure 4. Additional and alternative secondary struc-tures predicted using thermodynamic and comparativemethods. The left side of each panel shows the predictedsecondary structure, colored as in Figure 3, and the rightside of each panel shows the t to the correspondingregion in the cryo-EM density map.13(a) The L11-BD,where additional structure is predicted for the adjacentsequence (orange) using thermodynamic predictionsfrom mfold.36(b) The L1-BD, where additional pairingis predicted using mfold36for the rRNA sequence thatinteracts with MRP-L1 (red). A large globular mass ofunidentied protein(s) (marked by * on the semitran-sparent green density) may restrict the movement of theL1 region in the mitoribosome. (c) The domain III regionwhere an alternative secondary was predicted usingAlifold.34This structure differs from that predicted bycomparative sequence analysis (Figure 1(a)) and ts thecorresponding cryo-EM density much better (not shown).(d) The cryo-EM density for the large mitoribosomesubunit is shown in blue (interface view). The threeregions that have been modeled using additional andalternative folding predictions are shown in orange andlabeled a, b and c to correspond with the precedingpanels.200 Structural Evolution in Mitochondrial Ribosomes 9. the mitoribosome subunits. Interactions with thepenultimate stem of the small ribosomal subunit(SSU helix 44) are maintained near the core of thesubunit. The top of helix 44 contains the decodingcenter, where interactions between the mRNA andthe A-site tRNA are examined for delity,39andsignals for delity may be transmitted throughinteractions with domain IVof the LSU rRNA duringtranslation. It is not surprising that these interactionsare largely conserved, but more peripheral inter-actions at the subunit interface are replaced by newinteractions between MRPs in each subunit.13The central cavity of the 39 S subunit is highlyconserved, because several essential structures arepreserved in domain V (Figure 5, red), including thePTC. The 5S-BD is missing, in agreement with theloss of 5 S rRNA in the mitochondrial genome. Thehelices that radiate from the core of the structure tothe L1 arm are maintained, but are shorter than inbacterial ribsosomes, making interactions with theL1 protein in the mitoribosome unique. Thepositions of functional structures near the peripheryof the structure (L11-BD and SRL) are conserved,thereby preserving critical interactions with elonga-tion factors during the translation cycle.Protein homology modelingOf the 48 proteins found in the large subunit ofthe mitoribosome, 28 are homologous to prokar-yotic ribosomal proteins,8while the remaining 20are unique to the bovine mitoribosome. All of thebovine MRPs are encoded in the nuclear genome,9and must be translated in the cytoplasm andtransported into the mitochondrion.40,41MRPs aremuch larger than prokaryotic ribosomal proteins,and they often have insertions at the N terminus, atthe C terminus or both. It was originally thoughtthat the increase in protein size compensates forreduction in the mito-rRNA,33but the cryo-EMstructure does not support this theory,13as onlyw20% of deleted rRNA components are position-ally replaced by mitoribosome-specic proteins orextensions of homologous proteins.We compared all 48 MRP sequences68withsequences of ribosomal proteins whose structureshave been determined by X-ray crystallography.When signicant levels of identity and similaritywere found, we were able to generate proteinhomology models based on templates from X-raycrystal structures of ribosomal large subunitcomplexes.21,25,28,42We created partial models for16 proteins (Figure 6), and a summary of thosemodels is given in Table 1. (Note that L7 and L12 areidentical.)None of the ribosomal proteins could be modeledcompletely. Homology between the MRPs and theprokaryotic ribosomal proteins did not extendacross the entire length of the protein sequences.Commonly the N terminus, the C terminus, or bothtermini were unique. More detailed sequenceanalysis revealed that these regions are insertions,Figure 5. Three-dimensional model for the mitochondrial 16 S rRNA. (a) The 16 S rRNA is represented from theinterface and solvent-accessible sides of the structure and colored by domain (I, purple; II, dark blue; III, orange; IV,green; V, red; and VI, yellow). (b) The model RNA t to EM density that is attributable to RNA,13except for the tip of adomain V helix that contacts the L1 protein. However, the model of the extended rRNA segment ts into the completeLSU map (also see Figure 4(b)). Coloring is the same as in (a).Structural Evolution in Mitochondrial Ribosomes 201 10. not mutational differences. This trend has beendescribed,68and the results from our comparativestudy conrm those ndings.It had been proposed that these insertionsrepresent the addition of functional domains tocompensate for the decreased size of rRNA in thelarge subunit.68We analyzed these insertions todetermine if functional roles could be identiedfrom sequence homology to proteins of knownfunction. Unfortunately, the homology searchagainst the non-redundant (nr) database did notproduce any homology matches, suggesting thatthe sequence insertions in mammalian MRPs areunique.MRP structure renementThe homology models for the proteins were t tothe cryo-EM density in much the same way as theRNA helices. During protein tting, the mito-rRNAmodel (modeled again with the P-atom reducedrepresentation) was treated as a rigid scaffold thatwas held xed. Each protein (modeled withpseudoatoms centered on the Cacoordinates, calledC-atoms) was placed manually into the EM density,and rigid-body Monte Carlo with simulated anneal-ing was used to rene protein positions. Renementincluded VLAT terms for scoring the ts of theproteins to the density plus a set of restraints basedon conserved RNAprotein interactions observedin bacterial crystal structures. These interactionswere determined by measuring distances betweenevery nucleotide in the RNA and every amino acidfor each protein in the crystal structure of thebacterial ribosome. An interaction threshold of 3 Awas used for protein models that had homologousproteins in the H. marismortui structure (where all-atom detail is present),28whereas a threshold of 6 AFigure 6. Structural homology models for MRPs. (a) Models of the 16 MRPs with signicant sequence similarity tobacterial ribosomal proteins that have been structurally characterized by X-ray crystallography. (b) Structuralorganization of proteins in the large mitochondrial subunit t to the cryo-EM density (stereo view).202 Structural Evolution in Mitochondrial Ribosomes 11. was set for protein models with homologousstructures available from Thermus thermophilus21orDeinococcus radiodurans25(where all-atom detail isnot available). If the nucleotide and amino acidinvolved in the interaction were both conservedin the mitochondrion, harmonic restraints ofequivalent length were included between appro-priate P-atoms and C-atoms during renement.Of the 16 protein models, 13 had conservedinteractions with the RNA. The resulting RNAprotein restraints played a critical role in tetheringthe proteins to their conserved positions in themitoribosome model during renement, becausemuch of the protein density remains unlled, and itwould otherwise be difcult to guarantee that therenement would not move these proteins intoinappropriate regions of the density. The positionsof the three proteins that did not have conservedinteractions were rened, although a lower startingtemperature was required during the renementprotocol to prevent large movements. Also, forproteins with long extended loops, minimalrearrangements of some loops were required topreventsteric overlapbetween theproteinandrRNA.All 16 proteins are in positions very similar tothose found in the prokaryotic structures(Figure 6(b)). In most cases where restraints areavailable, the nucleotide-binding sites for themodeled proteins are conserved. For example, thebinding site for protein L2 is conserved in domainIV, which contains helices responsible for position-ing the protein in the same relative orientation as inthe bacterial ribosome. L11 and L7/12 are also inpositions very similar to those in bacteria, so thegeometries of their interactions with protein cofac-tors and the incoming A-site tRNA in mitochondriashould be similar to those in bacteria. However, therRNA-binding site for L1 (L1-BD) is very differentin the mito-rRNA than it is in bacterial rRNA. L1 isrepositioned further from the putative E-site, whichmay be absent from the mitoribosome.13,24Some ofthis difference is presumably due to the drastictruncation of the L1-BD in mito-rRNA, but it mayreect the greater exibility of the L1 arm of thelarge subunit2527when compared with nuclear-encoded ribosomes (see Discussion).The nal model of the LSU mitoribosomeThe nal model for the LSU of the mammalianmitoribosome ts the cryo-EM density nicely(Figure 7). We have placed 93% of the mito-rRNAsequence as well as 16 MRP models with signicantsequence similarity to prokaryotic ribosomalproteins. The L1-BD of the RNA that extends outof the density attributed to RNA does t the densityTable 1. Proteins modeled and their homologsProteinaHomologsbIdentity (%) Similarity (%) Model size (residues) MRP size (residues)MRP-L1 1GIY 28.6 52.4 100289 303MRP-L2 1NKW 38.2 56.6 126261 3051PNU 36.8 54.41GIY 40.4 57.41JJ2 31.6 51.5MRP-L3 1NKW 28.4 50.7 96306 3481PNU 28.4 51.1MRP-L4 1PNU 33.1 56.0 97271 311MRP-L7 1GIY 31.2 47.1 61198 198MRP-L12 1GIY 31.2 47.1 61198 198MRP-L11 1GIY 42.8 61.4 13157 1781NKW 35.2 57.21PNU 35.2 57.2MRP-L13 1NKW 29.1 57.4 1148 1781PNU 29.1 57.4MRP-L16 1NKW 27.1 54.2 71188 2511PNU 27.1 54.2MRP-L17 1NKW 33.6 59.5 11126 1751PNU 33.6 59.5MRP-L19 1NKW 26.5 48.0 96193 2801PNU 26.5 48.0MRP-L20 1NKW 36.4 63.6 8125 1491PNU 36.4 63.6MRP-L22 1GIY 29.2 47.5 69178 2061PNU 21.7 48.3MRP-L24 1PNU 35.4 56.3 59154 216MRP-L27 1NKW 43.5 65.2 3199 1481PNU 43.5 65.2MRP-L33 1NKW 51.9 71.2 960 651PNU 51.9 71.2MRP-L34 1NKW 44.7 65.8 5390 921PNU 44.7 65.8aMitochondrial ribosomal proteins (MRPs) with signicant homology to ribosomal proteins that were characterized previously arelisted.bThe homologs that were identied are listed by the Protein Data Bank accession code (1GIY is from Thermus thermophilus, 1NKW isfrom Deinococcus radiodurans, 1PNU is from Escherichia coli, and 1JJ2 is from Haloarcula marismortui).Structural Evolution in Mitochondrial Ribosomes 203 12. Figure 7. Stereo-view representation of the 3D model of the 39 S subunit of the mitochondrial ribosome. (a) Theinterface view of the subunit shows that the conserved interface of the mitochondrial ribosome is still dominated byrRNA structure (colored as in Figure 5). (b) The homologous MRPs (grey) are located predominantly towards thesolvent-accessible side of the particle. Upper and lower panels in both sections show the modeled structure (rRNA andproteins), and its tting into the cryo-EM map,13respectively.204 Structural Evolution in Mitochondrial Ribosomes 13. of the complete subunit, and the placement of theL1 protein suggests that an interaction between theRNA and protein is possible. Our placement ofthe L7/12 dimer is based on an earlier X-raycrystallographic study,21and the protein doesprotrude slightly from the EM density. Since thisregion is known to be exible in bacterial ribosomalparticles,43,44similar exibility in mitochondriacould explain the weaker density for the proteinin this region. Moreover, in a recent study45it hasbeen suggested that the traditionally assigned stalkof the LSU actually represents the protein L10 andmultiple copies of L7/12. If this scenario is true inthe mitoribosome also, our placement of L7/12would need further renement.DiscussionOur model of the large subunit of the mammalianmitoribosome suggests that the reduction in rRNAsize compared to that of bacteria does little to alterthe spatial organization of structural and functionaldomains common to the LSU of mammalianmitoribosomes and prokaryotic ribosomes. How-ever, some conserved interactions, including partsof the tRNA-binding sites, are absent in themitoribosome. The interactions between the largesubunit and tRNAs at the A-site, P-site and E-site ofthe bacterial ribosome have been characterized byX-ray crystallography studies.21Figure 8 comparesthe interactions predicted by our model with thoseobserved for bacterial ribosomes.rRNA segments interacting with the P-site arehighly conserved between mitochondrial and cyto-plasmic ribosomes, and all tRNA contacts with theLSU are preserved in the mitoribosome (Figure 8).The importance of positioning the acceptor stemcorrectly, which holds the nascent polypeptidechain, is evident from the rRNA sequence con-servation for regions interacting with the tRNA.Furthermore, additional contacts are made betweenthe tRNA and the mitoribosome at this positionduring mitochondrial translation by protein(s) inthe central protuberance (the so-called P-sitenger). The conserved protein models that we tto the EM density are not in positions to participatein these new interactions, so the P-site ngerinteraction can be attributed to extension(s) of oneof the 12 unmodeled bacterial homologs or, morelikely, to an MRP unique to mitochondrial trans-lation.Interactions at the A-site are also highly con-served (Figure 8), showing the importance ofpositioning the A-site and P-site tRNAs duringcatalysis of the peptidyltransferase reaction.18Contacts are maintained also between the highlyconserved rRNA helix 69 and the minor groove ofthe tRNA D-stem. But interactions between the ASFand the D-loop and T-loop of tRNAs in cytoplasmicribosomes21are lost because of sequence reductionin domain II of the LSU rRNA (Figure 1(b)). ThesetRNA loops exhibit a large degree of variation insize and content in mitochondria,46,47suggestingthat the rRNA and tRNA sequences that normallyinteract have co-evolved to accommodate structuralchanges. D-loop and T-loop structures are notsensed by the ribosome at the P-site, and newinteractions with the P-site nger are located at theT-stem. It has been suggested that reductions in thesize of D-stems, T-stems and loops in somemitochondrial tRNAs may cause a dramatic changein the preferred angle between the two arms ofthese tRNAs,46,48and transient electric birefrin-gence studies have supported this suggestion.49Our model of the LSU mitoribosome would permitthis kind of variability in tRNA conformation at theA-site, but it is not clear what, if any, affect thisdifference may play in translational delity.The ribosomal E-site is markedly different inmitoribosomes, as most of the rRNA regionsresponsible for interacting with the E-site tRNA inthe bacterial ribosome are absent from the mitor-ibosome (Figure 8).24The one interaction that mightbe structurally conserved occurs near the base of theL1 arm in the rRNA model. The sequence in thisregion is markedly different in the mito-rRNAcompared with prokaryotes, so any interactionwill be different, if not completely lost. Further-more, the observation that the tRNA binds stronglyin the P-site, but not in the E-site, suggests that theE-site is either very weak or non-existent in themitoribosome.13Many interactions between the prokaryotic 23 SrRNA and E-site tRNA involve contacts near theL1-BD.21These interactions and the exibility ofthe L1 arm (Figure 9(b), L1) suggest that E-siteoccupancy on the ribosome may be coupled toFigure 8. Interactions with thetRNA-binding sites of the mito-ribosome. The A-site, P-site andE-site tRNAs are represented withthe structure from yeast tRNA-Phe.76Interactions with rRNAsequence that were found in theX-ray crystal structure of theT. thermophilus 70 S particle21arerepresented as either beingconserved in (red) or absentfrom (yellow) the mitochondrialribosome.Structural Evolution in Mitochondrial Ribosomes 205 14. dynamic motions in this structure.2527The mito-chondrial L1 protein is readily identiable inthe EM density, interacting with the shortenedmitochondrial rRNA stem-loop in domain V(Figure 4(b)), but the protein is positioned farfrom the putative E-site (Figure 9(a)). While thisorientation may be due, in part, to the inherentexibility of the L1-BD, the truncation of the rRNAmay limit the range of motion. The EM densitycorresponding to L1 makes contacts with neighbor-ing, unassigned protein density (Figure 4(b),marked with *), which may further restrict itsmobility. Since the L1 protein is conserved in themitoribosome, it is probably important for somefunction, but it may not have a direct role in tRNAbinding.On the opposite side of the mitoribosomal LSU,the L11-BD is positioned by a unique structure(when compared to Archaea; Figure 9) because ofsequence reduction in the region that connects theconserved cofactor-binding domain and the core ofthe particle (Figure 3, orange region in domain II).Therefore, the spatial orientation of this conservedstructural domain near the periphery is conserved.The crystal structure of mitochondrial EF-Tu incomplex with GDP50is similar to homologousbacterial structures from Escherichia coli51andThermus aquaticus,52which would suggest that theoverall binding of cofactor with the ribosome inmitochondria is comparable to that in bacteria.Unique mito-rRNA structures (i.e. the sequenceleading to the L11-BD) may also be stabilized byadditional protein interactions found in the mitor-ibosome. The placement of 16 MRP models beginsthe assignment of structure to the increased proteinmass in the large subunit of the mitoribosome. Theunique MRP sequences suggest that their role intranslation is likely specic to mitochondria. Theunidentied protein densities are mostly localizedto the peripheral regions of the large subunit, andall of the proteins synthesized by mitoribosomes areinserted into the inner mitochondrial membrane.Therefore, unique MRPs appear to be associatedwith positioning the mitoribosome during cotran-slational insertion of nascent polypeptides into themembrane53and/or stabilizing extended rRNAstructures created by the large reductions insequence.The conservation of rRNA structure in bovinemitochondria does not correlate directly withsequence conservation. The overall base content isvery G-poor and A-rich when compared to archaealFigure 9. Three-dimensional models of mitochondrial and archaeal large ribosomal subunit rRNAs. Models are shownfrom the subunit interface side (left) and from the solvent side (right). (a) Mitochondrial rRNA, showing a dramaticreduction compared to archaea. (b) H. marismortui rRNA from X-ray crystallography.28The L1-arm (L1) was modeledusing structural data from other crystallographic structures.21,38The A-site nger (blue RNA helix adjacent to *) wasmodeled using sequence data and cryo-EM density from E. coli.64Six domains in both models (a) and (b) are identiedby different colors: I, purple; II, dark blue; III, orange; IV, green; V, red; and VI, light blue. The 5 S rRNA in the archaeon(yellow) is absent from the mitoribosome. L1 proteins for both models are shown with space-lling representations(grey).206 Structural Evolution in Mitochondrial Ribosomes 15. (H. marismortui) and bacterial (E. coli) species(Figure 10(a)). While guanine is the most frequentlyoccurring base in these prokaryotic rRNAs (30%), itis sharply reduced in bovine mitochondria (18%). Incontrast, the adenine content (25% in prokaryotes)jumps to 38% in the mitochondrion. The fraction ofnucleotides found in standard secondary structurebase-pairing drops from w60% in prokaryotes to45% in mitochondria (Figure 10(b)). This is partlydue to the reduction in guanine content without acorresponding reduction in cytosine content, sincethe latter have fewer prospective base-pairingpartners. The increase in adenine content alsocontributes to the decreased helical base-pairing inthe mitochondrial rRNA secondary structure(Figure 10(b)), because adenine bases pair lessfrequently than other bases. For example, 62% ofadenine bases are unpaired in the E. coli 16 S rRNA,while only 30% of G, C and U bases areunpaired.35,54An even larger fraction of adeninebases are unpaired in the E. coli 23 S rRNA(Figure 10(b)). The increased adenine content inthe mitoribosome results in reduced secondarystructure and facilitates formation of two uniquestructural features: (1) stretched regions thatreach across large distances to connect functionalregions whose positions are not changed (e.g. theL11-BD, whose position is maintained at theperiphery to interact with translation cofactors);and (2) large, unpaired loops at helix ends that mayassume globular structures and serve as recognitionelements for some of the MRP binding, e.g. theA-rich loops in domains II and III (grey in Figure 3).It is well known that adenine bases are involved in anumber of unusual tertiary structures,5560and theextended structures reported here are additionalexamples of such structures.It is not clear why evolutionary pressures lead tosuch a remarkable decrease in the size of the LSUmito-rRNA and a corresponding increase in proteincontent, but two structural principles have emergedfrom the present study. First, the key functionalsites occupy essentially the same positions as inprokaryotic ribosomes. Second, the signicantdecrease in G content and increase in A contentleads to a marked reduction in the fraction of base-paired nucleotides, yielding unique structures thatcan span large distances to maintain the 3Dorganization of structures essential for translation.MethodsCryo-electron microscopy and 3D imagereconstructionCryo-EM grids were prepared according to standardprocedures.61Data were collected on a Philips FEIFigure 10. Sequence compositionof LSU rRNAs from: Bos taurusmitochondrion, red; H. marismortui,blue; and E. coli, green. (a) Themitochondrial rRNA exhibits asignicant reduction in guanine(G) content and increase in adenine(A) content relative to H. maris-mortui (an archaeon) and E. coli (aeubacterium). (b) The base-pairedfractions for each species. Thefrequency of base-pairing is sub-stantially lower in the mitochon-drial ribosome, especially forcytosine.Structural Evolution in Mitochondrial Ribosomes 207 16. (Eindhoven, The Netherlands) Tecnai F20 eld emissiongun electron microscope, equipped with low-dose kit andan Oxford cryo-transfer holder, at a magnication of50,760!. A total of 194 micrographs were scanned on aZeiss atbed scanner (Z/I Imaging Corporation, Hunts-ville, AL), with a step size of 14 mm, corresponding to2.76 A on the object scale. The projection-matchingprocedure62within the SPIDER software63was used toobtain the 3D map. The previous 13.5 A resolution map ofthe 55 S mitoribosome13was used as the reference for thealignment of the data set. Initially, 217,908 images, sortedinto 54 groups according to defocus value (ranging from1.2 mm to 4.6 mm), were picked. Because of the problem ofconformational heterogeneity, we had to eliminate a largeportion of the data set in order to improve resolution.Only 79,516 images were retained, after manual screeningand removal of images from over-represented groupswithin 83 equi-spaced views of the ribosome and wereincluded in the nal 3D reconstruction. The resolution ofthe nal CTF-corrected 3D map, estimated using theFourier shell correlation with a cutoff value of 0.5,64was12.1 A (or 8.5 A by the 3s criterion65). The falloff of theFourier amplitudes toward higher spatial frequencies wascorrected as described.64RNA and protein components ofthe 55 S mitoribosome map were separated computation-ally using a method66based on differences in the densitydistribution of the two moieties, taking into account themolecular masses and contiguity constraints.Comparative sequence analysisThe rRNA sequences were aligned manually with thealignment editor AE2 (developed by T. Macke67). Thisprogram runs on SUN Microsystems computers on theSolaris operating system. Ribosomal RNA sequences arealigned by juxtapositioning the nucleotides that map tothe same elements in the secondary and tertiary structuremodels to the same columns in the alignment. The rRNAstructure models were predicted with covariation anal-ysis,35a method that identies a conserved set of base-pairings and helices in a group of aligned sequences. Thesecondary structure diagrams for the B. taurus mitochon-drial LSU rRNA were templated from previouslypredicted mammalian mitochondrial LSU rRNA struc-ture models,20and modied for unique features in theB. taurus structure. The structure diagrams were drawnwith the interactive secondary structure program XRNA(B. Weiser and H. Noller, University of California, SantaCruz).RNA homology modelingThe model for the mito-rRNA is based largely on thecrystal structure of the large subunit from H. marismortui(PDB accession code 1JJ2).28To begin, conserved RNAhelices were identied on the basis of the comparativesequence analysis. Having identied homologous regionsin the structure, simple changes could be made in thecases where a base-pair or an individual base (in a loop orbulge) could be changed to the corresponding nucleo-tide(s) found in the mitochondrial sequence (i.e. A-U pairchanged to C-G) using the Biopolymer module of theInsight-II software package (Molecular Simulations, Inc.,San Diego, CA). Other changes involve mutations thatresult in non-canonical base-pairing where normal base-pairs are found in the H. marismortui structure. In stillother cases, a canonical base-pair may be found inthe mitochondrial structure where a non-canonical pairexists in the archaeal ribosome. For these cases, the pairfound in the mitochondrial structure is superposed on thepair found in the H. marismortui structure. A round ofenergy minimization was used to satisfy the backbonegeometry while preserving the hydrogen bond inter-actions between the base-pairs.For regions where a greater difference is found betweenthe sequences, the mitochondrial rRNA was modeled onthe basis of previously characterized structures withsimilar or identical sequences.30These include commonmotifs found in RNA tertiary structures;68such astetraloops,55U-turns,69and so on. For structures that arenot as common, RNA structures previously determinedby X-ray crystallography are used as a library, ordatabase, for generating a 3D model. Sometimes,sequence comparisons suggested more than one possiblestructure. In such cases, ts to the cryo-EM density wereused to determine which candidate was more likely.Additional/alternate secondary structure predictionsThe B. taurus mitochondrial rRNA sequence was takenfrom the genomic sequence NC_001567 using the Entrezgenome database at NCBI.70Potential secondary struc-tures for regions of interest were predicted by mfold36and the Alifold34program in the Vienna RNA package.Default settings were used to predict secondary structurewith mfold. Elements of secondary structure that wereconsistent across several thermodynamic predictions,levels of lineage, or both, were selected as potentialsecondary structures. These were used to predict 3Dstructures that were validated or rejected by tting to thecryo-EM density.Multiple sequence alignments were created with themitochondrial LSU rRNAs from B. taurus and relatedorganisms (Bovinae, Bovidae, Pecora, Ruminantia,and Cetarteriodactyla), as dened the NCBI TaxonomyBrowser.70The sequences were obtained using Entrez(nucleotide or genome) and aligned using CLUSTALW.71Regions of interest were cut from each alignment andsubmitted to Alifold twice, once with a default covarianceweight of 1, and again with a covariance weight of 10. Inboth cases isolated base-pairs were allowed. Alternativesecondary structures were modeled in three dimensionsand then examined in the cryo-EM density map to selectthe one that t the best.Protein homology modelingThe sequences of all 48 proteins of the mitoribosome68were identied and searched against the non-redundantsequence database (nr) and the Protein Data Base (PDB)using the program BLASTP.72MRPs homologous toribosomal proteins in the same family were modeled forthose cases where the crystal structure has beendetermined. CLUSTALW71was used to create sequencealignments. We used the aligned sequences in themodeling program MODELLER673to create structuralmodels. Most of the templates selected for the modelingcontained only the Caatom of each amino acid residue,due to limited resolution of the crystal structures.Therefore, the remaining atomic positions were extrapo-lated using probability density functions.73This processcan sometimes lead to positioning that is unfavorable, sosome manual adjustments were made with the proteinusing Insight-II (Molecular Simulations, Inc., San Diego,CA). The models were optimized by steepest decentenergy minimization to remove any unfavorable bonds,208 Structural Evolution in Mitochondrial Ribosomes 17. angles and steric conicts. The structural characteristicsof the models were then examined with PROCHECK.38Regions that could not be modeled were further checkedagainst the nr and PDB databases to determine ifhomologous sequences or structures could be determinedusing just those short, unmodeled regions.Determining protein interactionsA simple Python script was written to determine theinteractions between RNA and proteins using variouscutoff distances, based on the resolution of thecrystal structure. This procedure works well for theH. marismortui structure,28because the all-atom detailallows the use of a 3 A cutoff to determine specichydrogen bonding pairs between RNA and proteinatoms. Not all mammalian MRPs have a high level ofhomology to proteins in the archaeal structure, so specicinteractions cannot be determined for all proteins, buthomologous proteins are also found in the crystalstructures from Thermus thermophilus21(PDB accessioncode: 1GIY), and D. radiodurans25(PDB accession code1NKW). These structures contain only Cacoordinates forthe proteins, and 1GIY provides only the phosphatepositions for each nucleotide. Therefore, PCadistanceswere measured using a 6 A cutoff for homologous proteinstructures in these eubacterial complexes, providing a listof neighboring residues between the RNA and protein.MRP-L11 is homologous to E. coli L11, so the all-atomcrystal structure of the L11-RNA fragment74(PDBaccession code 1KC8) was used to determine specicinteractions (3 A cutoff) between the RNA and protein.Proteinprotein interactions were analyzed using thesame approach.Once a list of interactions was determined for eachprotein model, restraints in the rigid-body Monte Carlorenement were applied to pairs of pseudoatomsdening interacting residues in both the proteins andnucleic acid. Of the 16 proteins, 13 have interactions withthe rRNA that could be expressed in such restraints, butL17, L19 and L24 did not have such interactions, due todeletions in the mitochondrial rRNA and MRP sequences.Structure renementCryo-EM density is incorporated as a structuralrestraint for renement of the model in YAMMP, our in-house molecular modeling package.32The vector lattice(VLAT) forceeld term denes the cryo-EM density as a3D potential, providing a score for the t of the model tothe density. Renement is done using the rigid bodyMonte Carlo module in YAMMP.The mitochondrial rRNAwas modeled using a reducedrepresentation with one pseudoatom per nucleotide.6Theinitial renement was performed by treating the entireRNA homology model as a rigid unit t to the completeribosome density, and it was subjected to 2!106steps ofMonte Carlo renement with simulated annealing as arigid body, starting at 1000 K with cooling to 10 K.A second round of renement was used to optimize thelocal t of each helix to the corresponding density. Eachhelix was treated as a separate rigid unit (52 units total,Figure 2), and renement was used to improve the totalVLAT score. To maintain connectivity between helicesalong the RNA chain, harmonic bonds were included inthe energy calculation as tethers between connectedhelices:Ebi Z kbibi Kbio2where Ebi denotes the energy of the ith bond; kbi is theforce constant for the ith bond; bi is the ith bond length;and bio is the corresponding equilibrium or ideal value.The ideal bond length is determined for each bond as thecrystallographic distance between consecutive nucleo-tides in two rigid units. The force constant was set at100 kcal/mol A 2for normal distances between con-secutive homologous nucleotides (usually 67 A ). Insome cases gaps were present due to sequences thatcould not be modeled (grey regions, Figure 2). A smallerforce constant (1 kcal/mol A 2) was used for these cases toallow more conformational freedom. A non-bond termwas also added to the energy calculation to preventinterpenetration of the helices:Eij Z kijrij Krijo2if rij %rijoEij Z 0 if rij Orijowhere Eij is the non-bond interaction energy betweenatoms i and j; kij is the non-bond force constant (100 kcal/mol A 2) for the atom pair ij; rij is the distance betweenatoms i and j; and rijo is the minimum distance allowedbetween the two atoms. The goal of the non-bond term isto provide volume exclusion so that double helices do notinterpenetrate, and a value of rijoZ7.5 A was used toachieve this.This second round of renement was performed usingmultiple rigid-body Monte Carlo with simulated anneal-ing. The starting temperature was set at 100 K, with a naltemperature of 10 K reached after 2!106steps. The nalposition of each rigid unit was accepted or rejected basedon the energy score and by visual inspection of the t withthe RNA-protein separated density using O.75The rRNAstructures were t to the complete subunit density, andthe regions corresponding to rRNA structures contained abetter score so the rened helices t the RNA density.Rened movements were rejected only when a helixshifted (a few angstrom units) allowing one strand to sitin the middle of the helical density (because the best twould rarely place phosphate groups at the boundary ofthe density). The structure was still associated with thecorrect density, but the small shift during renement wasnot accepted because the previous t placed thephosphate groups at the edge of the helical density,which t the density more accurately. Finally, the originalall-atom structures were superposed onto the renedphosphate positions and covalently linked, except for theregions where gaps occur. A nal round of energyminimization resolved small structural discrepanciescaused by structural rearrangements during the rene-ment protocol.Having placed the modeled rRNA structure in thedensity from EM, the proteins were then placed using asimilar protocol, with the RNA structure held in a xedposition. Each protein was treated as an independentrigid body (17 rigid bodies total, one RNA molecule and16 protein molecules). The overall placement of the L7/12dimer was based on the results of an earlier X-ray study,21and ts were done both as a dimer and as two monomers,to determine the structural organization that would bestagree with the density and the conserved interactionsbetween the two proteins. A consensus structure wasdetermined from both methods. Bonds were included inthe calculations using the previously determined PCa Documentation available at http://rumour.biology.gatech.eduStructural Evolution in Mitochondrial Ribosomes 209 18. distances for conserved residues between the RNA andprotein as the ideal bond length (bio) and a bond forceconstant (kb) of 10 kcal/mol A 2. The proteins were placedmanually in positions consistent with previous ribosomalcomplexes. Then 1!106steps of rigid body Monte Carlowith simulated annealing were performed over a range ofstarting temperatures (1000 K, 100 K, and 10 K) becauseof the varied restraints associated with each protein.Those with more restraints could sample conformationsat higher temperatures, while those with fewer restraintsrequired lower temperatures to prevent extensivemotions. All simulations were annealed to a naltemperature of 1 K. The nal position for each proteinstructure was evaluated both visually using O,75andquantitatively from the nal energy score.Protein Data Bank accession codeThe model has been deposited to the RCSB Protein DataBank with accession code 2FTC.AcknowledgementsWe thank Linda Spremulli for providing thesamples of mammalian mitoribosomes and JamieCannone for help with generating the secondarystructure diagrams. This work was funded bygrants from the National Institutes of Health toS.C.H. (GM53827), R.R.G. (GM67317) and R.K.A.(GM61576), and from the Human Frontier ScienceProgram to R.K.A. (RGY232003).References1. Attardi, G. (1985). Animal mitochondrial DNA: anextreme example of genetic economy. Int. Rev. Cytol.93, 93145.2. Chomyn, A., Cleeter, M. W., Ragan, C. I., Riley, M.,Doolittle, R. F. & Attardi, G. (1986). URF6, lastunidentied reading frame of human mtDNA,codes for an NADH dehydrogenase subunit. Science,234, 614618.3. Brenner, C. & Kroemer, G. (2000). 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