Ribosomal RNA

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Ribosomal RNADenis LJ Lafontaine, UniversiteLibre de Bruxelles, Brussels, Belgium

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

All proteins are synthesized by ribosomes, large RNA

protein complexes that functionas ribozymesand arethe targets of several clinically relevant antibiotics. Ribosomescontain

highly conserved rRNA species, which catalyse the key steps in protein synthesis, together

with 7080 proteins that play important roles in the correct folding and packaging

of the rRNAs.

Introduction

The ribosomal RNAs (rRNAs) lie at thecore of the proteinsynthesis machinery. These RNAs were long regarded asmere scaffolds for the ribosomal proteins (r-proteins) butrecent work has shown that the rRNAs in fact carryout thekey reactions in translation. A major function of the r-proteins is ensuring the correct structure of the rRNA,allowing its tight packing around the active centre of theribosome.

In all organisms the ribosome consists of two subunits.These are designated the 40S and 60S subunits ineukaryotes and the 30S and 50S subunit in Bacteria,Archaea and the cytoplasmic organelles of eukaryotes,mitochondriaand chloroplasts.In almost allorganismsthesmall ribosomalsubunit contains a singleRNA species (the18S rRNA in eukaryotes and the 16S rRNA elsewhere). InBacteria and Archaea, the large subunit contains tworRNA species (the 5S and 23S rRNAs); in most eukaryotes

the large subunit contains three RNA species (the 5S, 5.8Sand 25S/28S rRNAs). Sequence analysis shows that the5.8S rRNA corresponds to the 5end of the bacterial andarchaeal 23S rRNAs, and was presumably generated earlyin eukaryotic evolution by insertion of a spacer sequence.Chloroplast large ribosomal subunits also contain threeRNAs; in this case the 4.5S rRNA is derived from the 3 terminus of the bacterial 23S rRNA. Finally, in mitochon-dria the large subunit rRNA is smaller in size and isdesignated the 21S rRNA.

Organization of the Ribosomal RNAGenes

In almost all organisms the rRNAs are not synthesized assimple transcripts, but are generated from large precursors(pre-rRNAs) by posttranscriptional processing. In Bacter-ia and Archaea the primary transcript generally includesthe 16S, 23S and 5S rRNAs (see Figure 1a). These areflanked by the 5 and 3 external transcribed spacers (5-ETS and 3-ETS) and separated by the internal transcribed

spacer (ITS) regions. A transfer RNA (tRNA) gene igenerally located in the ITS between the 16S and 23rRNA genes, and one or more may also be located 3 to th5S gene. In eukaryotes, the 18S, 5.8S and 25/28S rRNAare cotranscribed by RNA polymerase I, while the 5S genis independently transcribed by RNA polymerase II

(Figure 1b,c).Most Bacteria and Archaea contain either a sing

rDNA operon or multiple copies of the operon dispersed ithe genome (for example, Escherichia colihas seven). Icontrast, eukaryotes generally have many copies of thrDNA organized in tandem repeats; in humans approximately 300400 rDNA repeats are present in five cluster(on chromosomes 13, 14, 15, 21 and 22). These sites aroften referred to as nucleolar organizer regions, reflectinthe fact that nucleoli were observed to assemble at theslocations in newly formed interphase nuclei. In thmajority of eukaryotes the 5S rRNA genes are present iseparate repeat arrays (Figure 1c). Unusually, in the yeas

Saccharomyces cerevisiae, a 5S rRNA gene is present ieach of the 100200 tandemly repeated rDNA repeats (ochromosome XII) (Figure 1b).

Article Contents

Introductory article

. Introduction

. Organization of the Ribosomal RNA Genes

. Pre-rRNA Processing

. rRNA Function

. rRNA and Phylogeny

16S tRNA(s)

23S (tRNA)

5S

30S

16S tRNA(s)

23S (tRNA)

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 Ribosomal DNA (rDNA) organization in different species.

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Pre-rRNA Processing

In all organisms, the mature rRNAs are generated byposttranscriptional processing reactions. In Bacteria andArchaea, the endonuclease RNAase III cleaves stemstructures formed by complementary sequences that flankeach of the mature rRNA sequences (see Figure 2a). The

separated pre-rRNAs are 3 processed by the 3 to 5exoribonuclease RNAase T and 5 processed by theendonuclease RNAase E. Processing occurs cotranscrip-tionally, but the requirement for the stem structures meansthateach mature rRNA must be fully synthesized before itsprocessing can commence. RNAase III also processesother RNA substrates, messenger RNAs (mRNAs) andphage and plasmid transcripts, while RNAase T partici-pates in the processing of tRNAs and other stable RNAs.

Pre-rRNA processing is less well understood in eukaryotes. In particular, many of the processing enzymeremain to be identified. Processing is posttranscriptionawith the exception of the initial cleavage by RNAase II(Rnt1p in yeast) in the 3-ETS which, at least in yeast, icotranscriptional. Subsequent processing shows a stron5!3bias in the order of cleavage. In yeast, processing o

the 18S rRNA involves four endonuclease cleavagewithin the 5-ETS and ITS1 and at the ends of the maturrRNA, but the endonucleases responsible have not beeidentified. ITS1 is also cleaved by an RNAproteicomplex, RNAase MRP. This is substantially homologouto RNAase P, that cleaves the 5ends of tRNAs. RNAasMRP cleavage allows entry for the 5 to 3 exonucleaseRat1p and Xrn1p that generate the 5 end of the 5.8rRNA. The 3 processing of the 5.8S rRNA is alsexonucleolytic and is carried out by a complex of eleve3to 5exonucleases called the exosome.

As in Bacteria, the eukaryotic pre-rRNA-processinenzymes also have additional substrates. Yeast Rnt1

(RNAase III) processes precursors to small nucleolaRNAs (snoRNAs) and spliceosomal small nuclear RNA(snRNA). The 5 to 3 exonucleases Rat1p and Xrn1degrade pre-rRNA spacer fragments and process the 5ends of snoRNAs, and Xrn1p degrades mRNAs 5to 3ithe cytoplasm. The exosome is involved in the degradatioof nuclear pre-mRNAs and pre-rRNA spacer fragmentthe processing of the 3 ends of snRNAs and snoRNAs ancytoplasmic mRNA turnover.

In both yeast and E. coli(the two organisms in whicRNA processing is best understood) RNA-processinenzymes are not specific to a single pathway, but have range of RNA substrates. The spacer regions of RNA

precursors change very rapidly during evolution anRNA-processing systems appear to have evolved to recruenzymes from a pool of factors as needed, rather than bdeveloping specific enzymes for each substrate.

Relationships between pre-rRNA processingin Bacteria and eukaryotes

There are extensive similarities in pre-rRNA processinacross evolution. The bacterial, archaeal and eukaryotipre-rRNAs are essentially colinear and several pre-rRNAprocessing enzymes are conserved from bacteria t

eukaryotes (Figure 2).

RNAase III

RNAase III is a protein endoribonuclease that cleaves botsides of imperfect double-stranded RNAs. In the bacteriapre-rRNA, RNAase III cleaves the stems that are formeby the sequences that flank the 16S and the 23S rRNAs (seFigure 2a), producing the substrates for subsequent trimming reactions. In the eukaryotic (yeast) pre-rRNA, th

16S RNA 23S RNA

tRNA

RNAase III RNAase III

30S pre-rRNA(a)

RNAase P

(b)

RNAase MRP

U318S RNA 5.8S RNA 25S RNA U8?

3 ETS

Rnt1p

ITS2ITS1A2

35S pre-rRNA

A3

A0 A1

5 ETS

A3A2

5.8S RNA 25S RNA

ITS25 ETS

ITS1

18S RNA

3 ETS

ITS2

18S RNA25S RNA5.8S RNA

Figure2 Pre-rRNAs in Escherichia coli(a)and Saccharomyces cerevisiae(b).Sites inthe E. colipre-rRNA that arecleaved by RNAaseIII andRNAase P areindicated. Sites in theS. cerevisiaepre-rRNA that are cleaved by Rnt1p and

RNAase MRP are also indicated. RNAase MRP is homologous to RNAase Pand Rnt1p is homologous to RNAase III. InE. coliprocessing at the ends of

the mature18S and23S rRNAs is coupled, since base pairing is required togenerate the RNAase III cleavage sites. In S. cerevisiaeit has been proposedthat interactionsin transwith the small nucleolar RNAs (snoRNAs) U3 and

U8 provides a similar coupling,although this has not yet been established.ETS, external transcribed spaces; ITS, internal transcribed spaces.

Ribosomal RNA

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homologue of RNAase III (Rnt1p) carries out the first stepin processing by cleaving a stem in the 3-ETS (seeFigure 2b).

RNAase MRP and RNAase P

RNAase MRP and RNAase P are endoribonucleases thatconsist of RNAprotein complexes. RNAase P processesthe5 ends of pre-tRNAs in all organisms and cleaves the 5end of the tRNA located in the ITS region in the bacterialand archaeal pre-rRNA (see Figures 1a and 2b). RNAaseMRP has only been identified in eukaryotes and in theeukaryotic (yeast) pre-rRNA RNAase MRP cuts withinITS1. The bacterial RNAase P consists of an RNAmolecule together with a single protein molecule. Incontrast, eukaryotic (yeast) RNAase P has nine proteincomponents together with a single RNA molecule. TheRNA components of RNAase P and MRP show simila-rities in their predicted structures and share eight proteincomponents; each also has one unique protein component.It is likely that RNAase MRP arose from RNAase P in anearly eukaryote and became specialized for pre-rRNAprocessing.

The RNA component of RNAase P from many Bacteriaand Archaea shows in vitro activity in the absence of itsprotein cofactor, although the salt conditions required forthis activity are far from physiological. An RNA moleculethat shows enzymatic activity in the absence of proteins istermed a ribozyme. It is believed that the RNA componentof bacterial RNAase P functions as a ribozyme in RNAcleavagein vivo. The protein component aids the bindingand release of the substrate, and allows cleavage of widerrange of substrates than with the RNA alone. In contrast,attempts to demonstrate ribozyme activity for the RNAcomponents of eukaryotic RNAase P or MRP have beenunsuccessful, suggesting that some or all of the enzymaticactivity has been taken over by the protein components.

Role of the small nucleolar RNAs in eukaryoticpre-rRNA processing

Eukaryotes contain a large number of snoRNA species(approximately 150 in human cells). With the exception ofthe RNA component of RNAase MRP, all of these speciescan be divided into two groups. These are designated as

box C1D and box H1ACA on the basis of conservedsequence elements that are believed to be sites of proteinbinding (Figure3). Each class of snoRNA is associatedwitha set of common proteins and members share conservedfeatures of predicted secondary structure. Most of thesesnoRNAs act as site-specific guides for the modification ofnucleotides within the rRNAs. The box C1D snoRNAsselect nucleotides at which the 2-hydroxyl positions of thesugar residues undergo methylation (2-O-methylation),whiletheboxH1ACAsnoRNAs select positions at which

uridine is converted into pseudouridine (C) by rotation othe base. SnoRNA homologues have recently beeidentified in Archaea where they also seem to be involvein rRNA modification.

In addition, a small number of box C1D and boH1ACA snoRNAs are required for pre-rRNA processing (U3, U14, snR10 and snR30 in yeast; U3, U8, U14 anU22in vertebrates). Genetic analyses showed that the yeasnoRNAs are allrequired for the pre-rRNA cleavage step

in the 5-ETS and ITS1, on the pathway of 18S rRNAsynthesis. In Xenopus (frog) oocytes, U3, U14 and U22 arsimilarly required for 18S rRNA synthesis, while U8 required for processing in the 3-ETS and in ITS1 on thpathway of 5.8S/28S rRNA synthesis. In the case of thyeast box C1D snoRNAs U3 and U14, compensatormutations have demonstrated that they must base paiwith the pre-rRNA to function in ribosome synthesis.

The requirement for these snoRNAs in processing doenot appear to be related to roles in rRNA modification anthe snoRNAs also do not appear to act catalytically iprocessing. Rather, they are thought to mediate changes ithe structure of the pre-rRNA, possibly establishing th

correct conformation for recognition by the endonuclease(s). In E. coli, the coordinated processing of the 5and 3 ends of the 16S and 23S rRNAs is ensured by threquirement that the flanking sequences base pair tgenerate the cleavage site for RNAase III (seeFigure2a).Nequivalent base pairing can be drawn for eukaryoteinstead it is speculated that interactions in trans witsnoRNAs brings the processing sites together and ensuretheir coordinated cleavage (see Figure2b). The U3 snoRNAis thought to provide this function for the coordination o

Box D Box C

Box C Box D

35pre-rRNA

2-OMe

2-OMe

snoRNA 5 3

Nop1p/fibrillarin

Nop58p

Nop56p

Snu13p

Cgf5p/Dyskerin

Gar1p

Nhp2p

Nop10p

3 5prerRNA

Box H Box ACA

snoRNA 5

N

CUGA

UGAU

GA

CUG

AAUGA

UGA

3

N

ANANNA ACA

Box C + D snoRNAsDirect 2-O-methylation

Box H + ACA snoRNAsDirect formation

Figure 3 The small nucleolar RNAs (snoRNAs) and their associatedproteins. The two major families of snoRNA are each associated with a

specific set of proteins. For the box C1D snoRNAs, these are Nop1p/fibrillarin, Nop58p, Nop56p and Snu13p. The H1ACA snoRNAs areassociated with Cbf5p/dyskerin, Gar1p, Nhp2p and Nop10p. Nop1p an

Cbf5pare the putative catalytic subunits. The colours of othercomponenof the two classes of snoRNP do not indicate functional homology.

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processing in the 5-ETS and ITS1, whileXenopusU8 maycoordinate processing in the 3-ETS and ITS1.

Structural rearrangements of the rRNAsduring ribosome synthesis

In the E. colipre-rRNA, the 5 end of the 16S rRNA isengaged in a base paired interaction with the 3region ofthe5-ETS. This interaction must be broken in order for the5end of 16S rRNA to assume its mature conformation this involves a long-range interaction between the loop ofthe5 stemloop structure and nucleotides around position917, an interaction referred to as the central pseudoknot.This structure is conserved throughout evolution and islikely to play a crucial role in the overall folding of therRNA. In the pre-rRNA of eukaryotes, the U3 snoRNAbase pairs to the 5stemloop structure of the 18S rRNA,preventing formation of the central pseudoknot. Forma-tion of the pseudoknot is presumably an irreversible stepand the alternative structure may block premature creationof this long-range interaction until the correct stage in theassembly process is reached. In yeast, a putative RNAhelicase (anenzyme that can open RNA structure) is foundstably associated with U3 and may catalyse this structuralisomerization.

In eukaryotes, large numbers of modification guidesnoRNAs base pair with the pre-rRNA. The conformationof the pre-rRNA in the snoRNA-associated form must bevery different from its mature structure, and extensivestructural rearrangements are inevitable. Remarkably, 17different putative RNA helicases that are required forribosome synthesis in yeast. These are likely to playessential roles in such structural remodelling.

It has been speculated that the primary function of themodification guide snoRNAs is to assist the correct foldingof the pre-rRNA. RNAs or proteins that function topromote the correct folding of another factor are termedchaperons. In this model, the rRNA modifications are aside product that serve as signals that the snoRNA hasbound (and has presumably done its work). Formation ofthe modification would trigger a helicase to dissociate thesnoRNA/pre-rRNA base pairing. There are, however, fewdata to support this model at present.

rRNA Function

Primary and secondary structure of rRNAs indifferent species

Inspection of the structure of rRNAs in distant organismsfrom the three domains of life reveals that despitesubstantial differences primary sequence, both the smallsubunit rRNA (SSU-rRNA) and large subunit rRNA(LSU-rRNA) display remarkable conservation of their

secondary, and probably tertiary, structures. Core structures can be drawn for the SSU- and LSU-rRNAs whiccan accommodate the secondary structures of all describerRNAs. The most conserved elements are presumed to bof functional significance. These have been proposed trepresent theactive core of the ribosome and the portion omodern ribosomes which was first established in the cours

of evolution. Notably, almost all the rRNA posttranscriptional modifications (base and ribose methylation as weas conversion of uridine to C) fall within these conservecore regions of the rRNAs.

Initial approaches to the structure of the rRNAinvolved the analysis of isolated fragments of the rRNAby chemical probing and RNARNA crosslinking experments. These analyses generated a great deal of data on thfunctional interactions within the ribosome but were lesinformative on the mechanisms of translation. A powerfutechnique for the determination of secondary structure wabased on phylogenetic comparisons. The principal approach is to look for compensatory mutations in region

predicted to base pair. Compensatory mutations ariswhen pairs of changesin sequence occursuchthat the basepair potential is retained with different nucleotides. Thidentification of multiple compensatory mutations ipredicted stem structures provides strong evidence fothe existence of actual base pairing. Backed by thcrosslinking data, this allowed the overall secondarstructure of the conserved regions of the rRNAs to bestablished with good confidence. The structures of botribosomal subunits from Archaea have recently beedetermined by X-ray crystallography, offering the firview of ribosome structure at atomic resolution. Thstructure has already helped to rationalize several decade

of biochemical andgenetic data. A major breakthrough fothe whole field of RNA biology is that the structurprovides decisive support for the view that the peptidyltransferase reaction (the reaction by which amino-aciresidues are attached to each other to form proteins) icatalysed by the rRNA itself.

In the primary sequences of the rRNAs, the conservesequences are separated by variable regions, in which thprimary and secondary structures diverge more rapidly ievolution. The overall length of these regions is also poorlconserved and is generally longer in eukaryotes; they artherefore often referredto as expansion segments. The corstructures of the SSU- and LSU-rRNAs contain 10 and 1

such variable regions, respectively. In general, the variablregions are more dispensable for ribosome function.

Functional domains in the rRNAs

The combination of biochemical approaches, mostlcrosslinking experiments and chemical footprinting orRNAs bound to tRNAs or to various antibiotics, with thgenetic analysis ofE. colistrains bearing mutations in the

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rRNAs led to the definition of several functional domainsin the rRNAs. As summarized in Table 1, domains wereascribed to the most basic functions of the ribosome (i.e.decoding or codonanticodon recognition and peptidyl-transferase activity) as well as to antibiotic binding andinteractions with ribosomal proteins and translationalfactors. The general picture which emerged from these

studies is that the decoding centre (the specific recognitionof the codon by the tRNA) of the ribosome lies within itssmall subunit and the peptidyltransferase activity (addi-tion of the new amino acid to the growing polypeptide) iscarried out by the large subunit. The accuracy oftranslation is determined by components of both subunits,probably reflecting interactions of the tRNAs with bothribosomal subunits.

The analysis of mutations in the rRNAs has long beenhindered by the large copy number of rDNA genes. InE.coli this problem was partially overcome by the over-expression of the mutated rDNA copy from a high copyplasmid construct providing a mixed rRNA population,

with about 50% each of the wild-type and mutantribosomes. In yeast, a number of more sophisticatedsystems for the conditional expression of mutant and wild-type rRNAs have been devised, allowing the analysis of theprocessing and function of the rRNA. These techniqueshave been extensively used for the analysis of the effects of

cis-acting mutations in the spacer regions on pre-rRNAprocessing. The effects of mutations on the function of thrRNA is less well characterized. However, it appears thafunctional domains, particularly the accuracy centre, awell as features of the rRNA that are recognized baminoglycoside antibiotics, have been highly conservethroughout evolution. Our understanding of the structur

and function of ribosomes in eukaryotes remains much lesadvanced than inE. coli.

Catalytic activities of the rRNAs duringtranslation

The identification of the ribosomal components involvein peptide-bond formation has been a longstandinchallenge in ribosome research. This area was mostlexplored in E. coli, making use of systems for in vitrreconstitution of the subunits.

Pioneering work used a simple peptidyltransferase (PTassay in which 50S subunits were mixed with twminisubstrates that mimicked tRNAs bound at the Aand P site (i.e. their structures resembled the tRNAcarrying the incoming, activated amino acid and the tRNAcarrying the elongating polypeptide chain, respectivelyallowing the formation of a single peptide bond. Thauthenticity of the reaction relied on the efficient inhibitio

Table 1 Functional domains within the E.colirRNAs

Functional domain Region of rRNA Major functions

Functionally relate

antibiotics

C1400 region 16S rRNA (14001500) DecodingTranslocation

Paromomycin

530 loop 16S rRNA (500545) Decoding

EF-Tu binding

Streptomycin

Helix 34 16S rRNA Decoding

EF-G function and translocation

Spectinomycin

912 region 16S rRNA (885912) Translational accuracy Streptomycin

Helix 45 (colicin fragment) 16S rRNA (14943end) ShineDalgarno interaction

Initiation factor binding

Subunit interface

Kasugamycin

Domain II (GTPase region) 23S rRNA EF-G-dependent GTP hydrolysis

ppGpp synthesis in stringent response

Thiostrepton

Micrococcin

Domain V (PT centre) 23S rRNA Peptide bond formation

Interaction with tRNA 3ends

Translational accuracy

Chloramphenicol

Erythromycin

Domain IV (1916 loop) 23S rRNA Interaction with anticodons and

tRNA 3ends

Translational accuracy

Subunit interface

sarcin loop 23S rRNA (26532667) EF-Tu and EF-G binding site Sarcin, ricin

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by PT-specific antibiotics such as chloramphenicol andcarbomycin. These early studies established that the PTactivity is not dependent on mRNA, 30S subunit,translational factors, guanosine triphosphate (GTP),adenosine triphosphate (ATP) or even intact tRNAs.Large ribosomal subunits lacking individual or multipleribosomal proteins were tested in this assay and showed to

be equally active. Only five proteins appeared to beessential for PT activity, two of which were shown to berequired for 50S subunit assembly. In a different set ofexperiments, ribosomes were subjected to harsh proteinextraction conditions. The particles were stripped nearly tocompletion but maintained their PT activity. Theseexperimental approaches did not determine whether thetwo or threeproteins that remained bound to the rRNAareinvolved in the PT activity per se or whether they arerequired to maintain a minimally competent rRNAstructure.

In the high resolution ( 3 A ) structure of the archaealLSU the peptidyl transferase (PT) region is seen to be

surrounded by a domain of tightly packed rRNA. Theproteins are generally located on the exterior of thisstructure, although some project into the rRNA domain,making extensive proteinRNA contacts and stabilizingthe tight structure of the rRNA around the catalytic activesite. These crystallographic studies also confirmed that PTactivity lies solely with the rRNA; no ribosomal proteinswere found within 18 A of the PTcentre (to have animpacton catalysis a protein would have to be within 3 A ). AnRNA-based mechanism for peptide bond formation hasbeen proposed involving the N3 position of an adenineresidue (A2251 inE. coli) and a charge relay system. Thisfirst activates the incoming peptide by accepting a proton,

and then neutralizes the charge on the leaving group afterpeptide bond formation. In essence, this is analogous to thereverse of the acylation step observed in serine proteases(e.g. chymotrypsin) during peptide hydrolysis and suggeststhat RNA have learned the chemical principles of catalysisbefore protein enzymes.

The current view is therefore that the catalytic activity ofthe ribosome lies in its RNA component while theribosomalproteins act as chaperones in ribosome assemblyand as cofactors to increase the efficiency of the RNA-mediated PT reaction and the accuracy of translation.

In vitro reconstitution of fully functional E. colismallribosomal subunits has long been achieved, either fromin

vitro transcribed or purified 16S rRNA and pools ofribosomal proteins. The situation was more complex forlarge ribosomal subunits, however. Active large ribosomalsubunits can only be reconstituted if an authentic fragmentof at least 80 nucleotides containing several posttranscrip-tionalmodifications is added intrans. This suggests that thefull activity of the large ribosomal subunit criticallyrequires one or more modified nucleotides.

Functional interactions between the rRNAs,mRNA and tRNAs

In Bacteria, the 3end of the 16S rRNA base pairs with thmRNA (see also Table 1). This is termed the ShineDalgarno interaction and is crucial for translation initiation. In all organisms, themRNA and tRNA interact in th

codonanticodon recognition which is reiterated throughout the whole translational process. This interactionwhich involves only three base-paired nucleotides, stabilized by a number of interactions between the tRNAand the rRNAs. Interactions important for the catalysis opeptide bond formation are provided by the recognition othe universally conserved 3CCA end of aminoacyl-tRNAsubstrates by 23S rRNA. In particular, a WatsonCricbase pair forms between a universally conserved residueG2252, close to the PT centre in the 23S rRNA and C74 athe acceptor end of tRNAs. In E. coli, an rRNAmRNAinteraction is proposed to play a role in the recognition othe termination triplet.

Interactions of the rRNAs with antibiotics

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

Most characterized antibiotics appear to bind primarilto the rRNAs. In some cases, the methylation status ospecific rRNA residues correlates with antibiotic resistancor sensitivity. For example, the N6-dimethylation of A205

in the 23S rRNA PT centre by the ErmE methyltransferasconfers resistance to lincosamide and streptogramin B anvariety of macrolide antibiotics, including erythromycinErythromycin binds to A2058 and neighbouring nucleotides, and mutation of these bases also confers antibiotiresistance. Similarly,resistance to thiostreptonis conferreby ribose methylation or substitution of A1067 in thGTPase region of the 23S rRNA, within the thiostreptonbinding site. However, both thiostrepton and erythromycin also make functionally important contacts witribosomal proteins (with L11 and L15, respectively). Icontrast, the N6-dimethylation of the A1518A1519doubleat the 3 end of 16S rRNA by the KsgA methylase i

required for sensitivity to kasugamycin. Atomic structureof ribosomal subunits bound to antibiotics have recentlbeen reported. These studies are starting to shed an entirelnew light on the interactions between antibiotics anrRNAs. For instance, several small subunit-specific antbiotics appear to exert their inhibitory effects through thstabilization of individualribosomal domains whichwoulotherwise show a degree of flexibility relative to each otheessential for decoding and possibly for the translocatioprocess.

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rRNA and Phylogeny

Highly conserved in structure and presumed functionacross all of evolution, the rRNAs, and particularly thesmall subunit rRNA, have become the most commonlyused markers for establishing phylogenetic relationshipsbetween organisms. Mutations in the conserved coreregions of the rRNA are heavily biased toward nucleotidesubstitution, rather than deletion and insertion. This,together with the existence of a universal secondary

structure, considerably facilitates the sequence alignmentprocess.

Molecular phylogeny (phylogeny derived from sequencecomparison) based on the rRNA sequence led to the

conclusion that there are three domains of life: Bacteri(previously termed Eubacteria), Archaea (previousltermed Archaebacteria) and Eukaryotes (also termeEukarya). Most models predict that during evolutiofrom the Progenote (the first organisms able to replicattheir genomes) the Archaea and eukaryotes arose from common ancestor, following their separation from th

bacterial line (see Figure 4). Trees with different topologhave been derived based on a variety of protein sequencesprobably as a consequence of the relatively high levels olateral gene transfer that is now thought to have occurreduring evolution.

Further Reading

Garrett RA, Douthwaite SR, Liljas A et al. (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: 679716.

Grosjean H and Benne R (eds) (1998) Modification and Editing of RNA

Washington, DC: ASM Press.

Hill WE,DahlbergA, Garrett RA etal. (1990) The Ribosome: Structur

Function and Evolution. Washington, DC: ASM Press.

Kressler D, Linder P and de la Cruz J (1999) Proteintrans-acting facto

involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol

cular and Cellular Biology 19: 78977912.

Lafontaine DL and Tollervey D (1998) Birth of the snoRNPs: th

evolution of the modification-guide snoRNAs.Trends in Biochemic

Science23: 383388.

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. 197219. New York

Cold Spring Harbor Laboratory Press.Puglisi JD, Blanchard SC and Green R (2000) Approaching translatio

at atomic resolution.Nature Structural Biology 7: 855861.

VenemaJ andTollervey D (1999) Ribosome synthesisin Saccharomyc

cerevisiae.Annual Review of Genetics 33: 261311.

Bacteria Archaea Eukaryotes

The tree of life - based onrRNA sequence comparison

Progenote

Figure 4 The tree of life. Universal phylogeny derived from comparisons

of rRNA sequences.

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