ARN Ribozomal

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Ribosomal RNADenis LJ Lafontaine, Institut de Biologie et de Me ´ decine Mole ´ culaires, Universite ´ Libre de

Bruxelles, Brussels, Belgium

David Tollervey, Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK

All proteins are synthesized by ribosomes, large ribonucleic acid (RNA) – proteincomplexes

that are the targets of several clinically relevant antibiotics. Ribosomes contain highly

conserved ribosomal RNA(rRNA)species, which catalyse the keysteps in protein synthesis,

together with 70 – 80 proteins that play important roles in the correct folding and

packaging of the rRNAs.

Ribosomal RNA

The ribosomal ribonucleic acids (rRNAs) lie at the core of the protein synthesis machinery. Recent results show thatthe rRNAs carry out the key reactions in protein synthesis.

The ribosomal proteins (r-proteins) play important, butancillary roles, which include ensuring the correctstructureof the rRNA and allowing its tight packing around theactive centre of the ribosome. See also: Ribosomalproteins: role in ribosomal functions; Ribosomal proteins:structure and evolution

In all organisms, the ribosome consists of two subunits.The small subunit is also designated the 40S subunit inEukaryotes andthe30Ssubunit in Bacteria andArchaea andin organelles – mitochondria and chloroplasts. The largesubunit is designated the 60S subunit in Eukaryotes and the50Ssubunit elsewhere. Theuse of a numberfollowedby ‘S’toidentify the ribosomal subunits and rRNAs reflects their ini-

tial characterization by velocity gradient centrifugation. In

almost all organisms, the small ribosomal subunit containssingle RNA species. This is called the 18S rRNA iEukaryotes and the 16S rRNA elsewhere, reflecting theisize differences. In Bacteria and Archaea, the large subuni

contains two rRNAspecies (the 5S and 23S rRNAs). In mosEukaryotes, the large subunit contains three rRNAs; twsmall species, the 5S, 5.8S rRNAs, and a large (>3 kb) species designated the 25S rRNA in yeast and the 28S rRNA ihigher eukaryotes. Sequence analysis shows that the 5.8rRNA corresponds to the 5’ end of thebacterialand archaea23SrRNAs, andwas presumably generatedearly ineukaryotic evolution by insertion of a spacer sequence. Chloroplalarge ribosomalsubunitsalso contain three RNAs,but inthcase the 4.5S rRNA is derived from the 3’ terminus of thbacterial23S rRNA. Finally,in mitochondria the single largsubunit rRNA is smaller in size and is designated the 21rRNA. See also: Archaeal ribosomes; Bacterial ribosome

Ribosome structure and shape

Article Contents

Introductory article

. Ribosomal RNA

. Organization of the Ribosomal RNA Genes

. Pre-rRNA Processing

. rRNA Function

. rRNA and Phylogeny

doi: 10.1038/npg.els.000383

16S t R N A ( s )

23S ( t R N A )

5S

30S

16S t R N A ( s )

23S ( t R N A )

5S

30S

(a) Escherichia coli

18S 25S 5S

35S

5.8S 18S 25S 5S

35S

5.8S

(b) Yeast

pol I pol I

pol III pol III

18S 25S

45S

5.8S 18S 25S

45S

5.8S

(c) Mammalian

5S (cluster)+

5S

Figure 1 rDNA organization in different species.

ENCYCLOPEDIA OF LIFE SCIENCES & 2006, John Wiley & Sons, Ltd. www.els.net



Organization of the Ribosomal RNAGenes

The rRNAs are not synthesized as simple transcripts, butare generated from large precursors (pre-rRNAs) by post-transcriptional processing. In Bacteria and Archaea, the

primary transcript generally includes the 16S, 23S and 5SrRNAs (see Figure 1a). These are flanked by the 5’ and 3’external transcribed spacers (5’-ETS and 3’-ETS) and sep-arated by the internal transcribed spacer (ITS) regions. Atransfer RNA (tRNA) gene is generally located in the ITSbetween the 16S and 23S rRNA genes, and one or moretRNA may also be located3’ to the5S gene. In Eukaryotes,the 18S, 5.8S and 25/28S rRNAs are cotranscribed byRNA polymerase I, while the 5S gene is independentlytranscribed by RNA polymerase III (Figure 1b and c).See also: RNA polymerases: subunits and functionaldomains; RNA synthesis; Transfer RNA synthesis andregulation

Most Bacteria and Archaea contain either a single rib-osomal deoxyribonucleic acid (rDNA) gene or multiplecopies that are dispersed in the genome (e.g. Escherichiacoli has seven). In contrast, Eukaryotes generally havemany copies of the rDNA organized in tandem repeats; inhumans, approximately 300–400 rDNA repeats are pre-sent in five clusters (onchromosomes 13, 14, 15, 21 and 22).These sites are often referred to as nucleolar organizer re-gions, reflecting the fact that nucleoli were observed toassemble at these locations in newly formed interphasenuclei. In the majority of Eukaryotes, the 5S rRNA genesare present in separate repeat arrays (Figure 1c). Unusually,in the yeast Saccharomyces cerevisiae, a 5S rRNA gene is

present in each of the 100–200 tandemly repeated rDNArepeats (on chromosome XII) (Figure 1b). See also:Bacterial ribosomes: assembly; Eukaryotic ribosomes:assembly; Repetitive DNA: evolution

Pre-rRNA Processing

In all organisms, the mature rRNAs are generatedby post-transcriptional processing reactions. There are extensivesimilarities in pre-rRNA processing across evolution. Thebacterial, archaeal and eukaryotic pre-rRNAs are essen-tially co-linear and several pre-rRNA processing enzymes

are conserved across evolution (Figure 2). However, thecomplexity of the ribosome synthesis pathway is muchgreater in Eukaryotes than in Bacteria. In excess of 200proteins are currently known to function in the post-tran-scriptional steps of ribosome synthesis in yeast, about 10-fold more than the number of known ribosome synthesisfactors in E. coli , and the complexity is unlikely to be lowerin human cells.

In Bacteria and Archaea, the endonuclease ribonuc-lease (RNase) III cleaves stem structures formed by

complementary sequences that flank each of the maturrRNA sequences (see Figure 2a). The separated pre-rRNAare 3’ processed by the 3’ to 5’ exoribonuclease RNase and 5’ processed by the endonuclease RNase E. Processinoccurs cotranscriptionally, but the requirement for thstem structures means that each mature rRNA must bfully synthesized before its processing can commencSee also: Messenger RNA in prokaryotes; Ribonuclease

Pre-rRNA processing in Eukaryotes is largely posttranscriptional, with the exception of the initial cleavage bRNase III (Rnt1p in yeast) in the 3’-ETS which, at least i

yeast, is cotranscriptional. Subsequent processing shows strong 5’!3’ bias in the order of cleavage. In yeasprocessing of the 18S rRNA involves four endonucleascleavages, within the 5’-ETS and ITS1 and at the ends othe mature rRNA, but the endonucleases responsible havnot been identified. ITS1 is cleaved by an RNA–proteicomplex, endoribonuclease MRP (RNase MRP). This isubstantially homologous to ribonuclease P (RNase Pthat cleaves the 5’ ends of tRNAs. RNase MRP cleavagallows entry for the 5’ –3’ exonucleases Rat1p and Xrn1

16S RNA 23S RNA

tRNA

RNase III RNase III

30S pre-rRNA(a)

RNaseP

(b)

RNase MRP

U318S RNA 5.8S RNA 25S RNA U8?

3’ ETS

Rnt1p

ITS2ITS1 A2

35S pre-rRNA

A3

A0 A1

5’ ETS

A3 A2

5.8S RNA 25S RNA

ITS25’ ETS

ITS1

18S RNA

3’ ETS

ITS2

18S RNA25S RNA5.8S RNA

Figure2 Pre-rRNAs in E. coli (a)and S. cerevisiae (b). Sites in the E.coli pr

rRNA that arecleaved by RNase III andRNaseP areindicated. Sites in theCerevisiae pre-rRNA that are cleaved by Rnt1p and RNase MRP are alsoindicated. RNase MRPis homologous to RNase P andRnt1pis homologo

to RNase III. In E. coli , processing at the ends of the mature 18S and 23SrRNAs is coupled, since base pairing is required to generate the RNase II

cleavagesites. In S. cerevisiae , it has beenproposedthat interactions in tra

with thesnoRNAs U3 andU8 provides a similar coupling,althoughthis h

not yet been established.

Ribosomal RNA

2



that generate the 5’ end of the 5.8S rRNA. 3’ processing of the 5.8S rRNA is also exonucleolytic and is carried outby a complex of 11 3’ –5’ exonucleases called the exosome.See also: Exonucleases; Messenger RNA in eukaryotes

RNase III

RNase III is a protein endoribonuclease that cleaves bothsides of imperfect double-stranded RNAs. In the bacterialpre-rRNA, RNase III cleaves the stems that are formed bythe sequences that flank the 16S and the 23S rRNAs (seeFigure 2a), producing the substrates for subsequent trim-ming reactions. In the eukaryotic (yeast) pre-rRNA, thehomologue of RNase III (Rnt1p) carries out the first stepinprocessing by cleaving a stem in the 3’-ETS (see Figure 2b).

RNase MRP and RNase P

RNase MRP and RNase P are endoribonucleases that

consist of RNA–protein complexes. RNase P processes the5’ ends of pre-tRNAs inall organisms and cleaves the 5’ endof the tRNA located in the ITS region in the bacterial andarchaeal pre-rRNA (see Figures 1a and 2b). RNase MRPhas only been identified in Eukaryotes and in the eukaryo-tic (yeast) pre-rRNA RNase MRP cuts within ITS1. Thebacterial RNase P consists of an RNA molecule togetherwith a single protein molecule. In contrast, eukaryotic(yeast) RNase P has nine protein components togetherwith a single RNA molecule. The RNA components of RNase P and MRP show similarities in their predictedstructures and share eight protein components; each alsohas one unique protein component. It is likely that RNase

MRP arose from RNase P in an early Eukaryote and be-came specialized for pre-rRNA processing. See also:Protein–RNA interactions; Transfer RNA modification

Role of the small nucleolar RNAs in Eukaryoticpre-rRNA processing

Eukaryotes contain a large number of small nucleolarRNA (snoRNA) species (approximately 150 in humancells). With the exception of the RNA component of RNa-se MRP, all of these species canbe divided into two groups.These are designated as ‘box C+D’ and ‘box H+ACA’ onthe basis of conserved sequence elements that are believed

to be sites of protein binding (Figure 3). Each class of sno-RNA isassociated with a set of common proteins and shareconserved structures. Most of these snoRNAs act as site-specific guides for the modification of nucleotides withinthe rRNAs. The box C+D snoRNAs select nucleotides atwhich the 2’-hydroxyl positions of the sugar residues un-dergo methylation (2’-O-methylation), while the boxH+ACA snoRNAs select positions at which uridine isconverted into pseudouridine (C) by rotation of the base.Homologues of the snoRNAs have also been identified in

Archaea where they also seem to be involved in rRNAmodification. See also: snoRNAs: biogenesis, structurand function

In addition, a small number of box C+D and boH+ACA snoRNAs are required for pre-rRNA processin(U3,U14,snR10andsnR30inyeast;U3,U8,U14andU2invertebrates). In the case of the yeast box C+D snoRNAU3 and U14, compensatory mutations have demonstratethat they must base pair with the pre-rRNA to function iribosome synthesis. These snoRNAs are thought to mediate changes in the structure of the pre-rRNA, possibly establishing the correct conformation for recognition by th

endonuclease(s). In E. coli , the coordinated processing othe 5’ and 3’ ends of the 16S and 23S rRNAs is ensured bthe requirement that the flanking sequences base pair tgenerate the cleavage site for RNase III (see Figure 2a). Nequivalent base pairing can be drawn for Eukaryotes; instead it is speculated that interactions in trans with snoRNAs brings the processing sites together and ensuretheir coordinated cleavage (see Figure 2b).

rRNA Function

Primary and secondary structure of rRNAs indifferent species

Inspection of the structure of rRNAs in distant organismfromthe threedomains of life (see the section onrRNAanphylogeny) reveals that despite substantial differences iprimary sequence, both the small subunit rRNA (SSUrRNA) and large subunit rRNA (LSU-rRNA), displaremarkable conservation of their secondary, and probablytertiary structures. Core structures can be drawn for th

Box D’ Box C’

Box C Box D

3’

5’ pre-rRNA

2’-OMe

2’-OMe

snoRNA 5’ 3’

Nop1p/fibrillarin

Nop58p

Nop56p

Snu13p

Cbf5p/Dyskerin

Gar1p

Nhp2p

Nop10p

3’ 5’ pre-rRNA

Box H Box ACA

snoRNA 5’

N

C U G

A U G A U

G A

C U G A A U G

A U G A

3’

N

ANANNA ACA

Box C + D snoRNAsDirect 2’-O-methylation

Box H + ACA snoRNAsDirect formation

Figure 3 The small nucleolar RNAs (snoRNA) and their associated

proteins. The two major families of snoRNA are each associated with aspecific set of proteins. For the box C+D snoRNAs, these are Nop1p/

fibrillarin, Nop58p, Nop56p and Snu13p. The H+ACA snoRNAs areassociated with Cbf5p/dyskerin, Gar1p, Nhp2p and Nop10p. Nop1p anCbf5p are the putative catalytic subunits.

Ribosomal RNA



SSU- and LSU-rRNAs which can accommodate the pre-dicted secondary structures of all rRNAs analysed. Themost conserved elements are presumed to be of particularfunctional significance. These are likely to represent anactive core regionthat was establishedearlyin the course of evolution. Notably, almost all post-transcriptional mod-ification of the rRNA (see the subsections on Role of the

small nucleolar RNAs in Eukaryotic pre-rRNA processingand Interactions of the rRNAs with antibiotics) occurswithin these conserved core regions of the rRNAs. Thesemodifications are believed to ‘fine tune’ the folding of therRNAs to achieve the very precise three-dimensional struc-tures that are required for their functions in ribosome syn-thesis. The conserved core sequences are separated byvariable regions, in which the primary and secondarystructures diverge more rapidly in evolution. In general,the variable regions are more dispensable for ribosomefunction. These regions are generally longer in Eukaryotesand they are therefore often referred to as expansion seg-ments. See also: rRNA structure

The structures of the small and large ribosomal subunitsand the intact 70S ribosome from Archaea have been de-termined by X-ray crystallography, offering the view of ribosome structure at atomic resolution. A major break-through for the whole field of RNA biology was that thestructure provided decisive support for the view that thepeptidyl transferase (PT) reaction (the reaction by whichamino acid residues are attached to each other to formproteins) is catalysed by the rRNA itself. See also:Phylogeny based on 16S rRNA/DNA

Functional domains in the rRNAs

As summarized in Table 1, the basic functions of the ribosome can be attributed to specific domains of the rRNAdecoding or codon–anticodon recognition and PT activityinteractions with r-proteins and translational factors awell as antibiotic binding. The decoding centre (the specifi

recognition of the codon by the tRNA) of the ribosome liewithin its small subunit, whereas the PT activity (additioof the new amino acid to the growing polypeptide) is carried out by the large subunit. The accuracy of translationdetermined by components of both subunits, reflectininteractions of the tRNAs with both ribosomal subunitSee also: Gene expression: decoding and accuracyTransfer RNA

Our understanding of the structure and function oribosomes in Eukaryotes remains much less advanced thain E. coli . However, it appears that functional domainsparticularly the accuracy centre as well as features of thrRNA that are recognized by aminoglycoside antibiotics

have been highly conserved throughout evolution.

Catalytic activities of the rRNA duringtranslation

The identification of the ribosomal components involvein peptide bond formation has been a long-standing chalenge in ribosome research. This area was mostly explorein E. coli , making use of systems for in vitro reconstitutioof the subunits.

Table 1 Functional domains within the E. coli rRNAs

Functional domain Region of rRNA Major functions Functionally antibiotic

C1400 region 16S rRNA (1400–1500) Decoding translocation Paromomycin

530 loop 16S rRNA (500–545) Decoding EF–Tu binding Streptomycin

Helix 34 16S rRNA Decoding EF–G function and

translocation

Spectinomycin

912 region 16S rRNA (885–912) Translational accuracy Streptomycin

Helix 45 (colicin fragment) 16S rRNA (1494–3’end) Shine–Dalgarno interaction Kasugamycin

Initiation factor binding

Subunit interfaceDomain II (GTPase region) 23S rRNA EF–G-dependent GTP hydrolysis Thiostrepton

ppGpp synthesis in stringent re-sponse Micrococcin

Domain V (PT centre) 23S rRNA Peptide bound formation Chloramphenicol

Interaction with tRNA 3’ ends Erythromycin

Translational accuracy

Domain IV (1916 loop) 23S rRNA Interaction with anticodons andtRNA 3’ ends

Translational accuracy

Subunit interfacea Sarcin loop 23S rRNA (2653–2667) EF –Tu and EF –G binding site Sarcin, ricin

Ribosomal RNA

4



Pioneering work using a simplePT assay established thatthe PT activity is not dependent on messenger RNA(mRNA), 30S subunit, translational factors, guanosinetriphosphate (GTP), adenosine triphosphate (ATP) oreven intact tRNAs. Large ribosomal subunits lacking in-dividual or multiple r-proteins were tested in this assay andshowed to be equally active. These analyses indicted that

the activity was likely to reside in the rRNA itself. This wasconfirmed in the high-resolution ( 3 A ˚ ) structure of thearchaeal LSU. The PT region is seen to be surrounded by adomain of tightly packed rRNA. The proteins are gener-ally located on the exterior of this structure, although someproject into the rRNA domain, making extensive protein– RNA contacts and stabilizing the tight structure of therRNA around the catalytic active site. No r-proteins werefound within 18 A ˚ of the PT centre (to have an impact oncatalysis a protein would have to be within 3 A ˚ ). The pep-tidyl-transfer reaction is energetically favourable and it iscurrently believed that the catalytic activity of the PT cen-tre derives primarily from the precise spatial positioning of

the A- and P-site tRNAs by the rRNA. See also: Lifelinesbeyond the genome: why chickens come before eggs;Peptidyl transfer on the ribosome; Peptide chain elonga-tion: models of the elongation cycle

The catalytic activity of the ribosome therefore lies in itsRNA component, while the r-proteins act as chaperones inribosome assembly and as cofactors to increase the effi-ciency of the RNA-mediated PT reaction and the accuracyof translation.

Functional interactions between the rRNAs,mRNA and tRNAs

In Bacteria, the 3’ end of the 16S rRNA base pairs with themRNA (see also Table 1). This is termed the Shine–Da-lgarno interaction and is crucial for translation initiation.At initiation, the Shine–Dalgarno sequence is bound in alarge cleft in the 30S structure, while some 30 nucleotides of the mRNA are bound around the neck of the 30S subunit.Eight nucleotides, centred on the A- and P-site codons, areexposed at the interface between the ribosomal subunits. Apronounced kink between the two codons allows the si-multaneous base pairing with the A- and P-site tRNAs.The codons are associated with the ‘decoding site’ region,near the 3’ end of the 16S rRNA, binding in the major

groove of a noncanonical helical structure. The interac-tions between the P-site tRNA and the small subunits aredependent on the 16S rRNA.

The mRNA and tRNA interact in the codon/anticodoninteraction, which involves only three base-paired nucleo-tides. A long-standing conceptual problem was the abilityof the translation machinery to distinguish between thecognate tRNA (i.e. the species that correctly matches thecodon in the mRNA) and near-cognate species with mis-matchesthatarepredictedtoresultinonlyasmallreduction

in the energetics of the interaction. Structural analyses indicate that the geometry of the codon–anticodon base pairis monitored, not just the overall affinity. This is sensed ban interaction between three bases of the 16S rRNA andthminor groove of the first two codon–anticodon base pairsThisshape-sensing greatly enhances the energetic differencbetween cognate and near-cognate interactions. The correc

base pairing triggers a structural change in the 30S subunand its closure around the cognate tRNA.

Interactions important for the catalysis of peptide bonformation are provided by the recognition of the universally conserved 3’ CCA end of aminoacyl tRNA substrateby 23S rRNA. In particular, a Watson–Crick base paiforms between a universally conserved residue, G2252close to the PT-centre in the 23S rRNA and C74 at thacceptor end of tRNAs. In E. coli , an rRNA–mRNA interaction is proposed to play a role in the recognition otermination codons. See also: Messenger RNA in prokaryotes; Messenger RNA: interaction with ribosomeProtein synthesis initiation in prokaryotes; Translationa

components in prokaryotes: genetics and regulation

Interactions of the rRNAs with antibiotics

Antibiotics have been of great use in ribosome researchparticularly where their site(s) of interaction with the ribosome could be correlated with specific translational defects, providing putative functions for particular sectionof rRNAs (see also Table 1).

Most characterized antibiotics appear to bind primarilto the rRNAs, although some functionally important interactions are made to r-proteins. In some cases, the methylation status of specific rRNA residues correlates wit

antibiotic-resistance or sensitivity. Atomic structures oribosomal subunits bound to antibiotics revealed details othe interactions between antibiotics and rRNAs. Severasmall subunit-specific antibiotics appear to exert their inhibitory effects throughthe stabilizationof domains, whicwould otherwise show a degree of flexibility relative to eacother that is essential for decoding and possibly for thtranslocation process. Seealso: Antimicrobial agents:basiof action; Bacterial antibiotic resistance

rRNA and Phylogeny

Highly conserved in structure and presumed functioacross all of evolution, the rRNAs have become the moscommonly used marker for establishing phylogenetic relationships between organisms. Mutations in the conserved core regions of the rRNA are heavily biased towardnucleotide substitution, rather than deletion and insertionThis, together with the existence of a universal secondarstructure, considerably facilitates the sequence alignmenprocess.

Ribosomal RNA



Molecular phylogeny (phylogeny derived from sequencecomparison) based on the rRNA sequence led to the con-clusion that there are three domains of life: Bacteria (pre-viously termed Eubacteria), Archaea (previously termedArchaebacteria) and Eukaryotes (also termed Eukarya).Most models predict that during evolution from the Pro-genote (the first organisms able to replicate their genomes)the Archaea and Eukaryotes arose from a common ances-tor, following their separation from the Bacterial line

(see Figure 4). Trees with different topology have been derivedbased on a varietyof protein sequences, probablyasconsequence of the relatively high levels of lateral gentransfer that is now thought to have occurred during evolution. See also: Molecular phylogeny reconstructionUniversal tree of life

Further Reading

Fromont-Racine M, Senger B, Saveanu C and Fasiolo F (2003) Ribo

some assembly in eukaryotes. Gene 313: 17–42.

Garrett RA, Douthwaite SR, Liljas A et al. (eds) (2000) The Ribosom

Structure, Function, Antibiotics and Cellular Interactions. Washingto

DC: ASM Press.

Green R and Noller HF (1997) Ribosomes and translation. Annu

Review of Biochemistry 66: 679–716.

Kiss T (2002) Small nucleolar RNAs: an abundant group of noncodin

RNAs with diverse cellular functions. Cell 109: 145–148.

Noller F (1999) On the origins of the ribosome: Coevolution of th

subdomains of tRNA and rRNA. In: Gesteland RF, Cech TR an

Atkins JF (eds). The RNA World , 2nd edn, pp. 197–219. New Yor

Cold Spring Harbor Laboratory Press.

Noller HF, Hoang L and Fredrick K (2005) The 30S ribosomal P site:

function of 16S rRNA. FEBS Letters: 579: 855–858.

Ogle JM, Carter AP and Ramakrishnan V (2003) Insights into the d

coding mechanism from recent ribosome structures. Trends in Bio

chemical Sciences: 28: 259–266.

VenemaJ andTollervey D (1999)Ribosome synthesisin Saccharomyc

cerevisiae. Annual Review of Genetics 33: 261–311.

Yusupova GZ, Yusupov MM, Cate JH and Noller HF (2001) The pat

of messenger RNA through the ribosome. Cell 106: 233–241.

Bacteria Archaea Eukaryotes

The tree of life - based onrRNA sequence comparison

Progenote

Figure 4 The tree of life. Universal phylogeny derived from comparisonsof rRNA sequences.

Ribosomal RNA

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