stop codon recognition and interactions with peptide release factor rf3 of truncated and chimeric...

Molecular Microbiology (2003)

50

(5), 1467–1476 doi:10.1046/j.1365-2958.2003.03799.x

© 2003 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology1365-2958Blackwell Publishing Ltd, 200350

514671476

Original Article

L. Mora, A. Zavialov, M. Ehrenberg and R. H. BuckinghamInteractions of truncated and chimeric RFs

Accepted 28 August, 2003. *For correspondence. [email protected]; Tel. (

+

33) 1 58 41 51 20; Fax (

+

33) 158 41 50 20.

Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from

Escherichia coli

Liliana Mora,

1

Andrei Zavialov,

2

Måns Ehrenberg

2

and Richard H. Buckingham

1

*

1

UPR9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 75005, France.

2

Department of Cell and Molecular Biology, BMC, Box 596, S-75124 Uppsala, Sweden.

Summary

Release factors RF1 and RF2 recognize stop codonspresent at the A-site of the ribosome and activatehydrolysis of peptidyl-tRNA to release the peptidechain. Interactions with RF3, a ribosome-dependentGTPase, then initiate a series of reactions that accel-erate the dissociation of RF1 or RF2 and their recy-cling between ribosomes. Two regions of

Escherichiacoli

RF1 and RF2 were identified previously asinvolved in stop codon recognition and peptidyl-tRNAhydrolysis. We show here that removing the N-terminal domain of RF1 or RF2 or exchanging thisdomain between the two factors does not affect RFspecificity but has different effects on the activity ofRF1 and RF2: truncated RF1 remains highly activeand able to support rapid cell growth, whereas cellswith truncated RF2 grow only poorly. Transplanting aloop of 13 amino acid residues from RF2 to RF1switches the stop codon specificity. The interaction ofthe truncated factors with RF3 on the ribosome isdefective: they fail to stimulate guanine nucleotideexchange on RF3, recycling is not stimulated by RF3,and nucleotide-free RF3 fails to stabilize the bindingof RF1 or RF2 to the ribosome. However, the N-termi-nal domain seems not to be required for the expulsionof RF1 or RF2 by RF3:GTP.

Introduction

Translation termination, the release of the newly synthe-sized polypeptide chain, occurs when the ribosome

encounters a stop codon on mRNA in its current readingframe (for reviews, see Kisselev and Buckingham, 2000;Nakamura

et al

., 2000; Poole and Tate, 2000; Kisselev

et al

., 2003). Eubacteria and eukaryotes use the threestop codons UAG, UAA and UGA. In eubacteria, two class1 peptide release factors (RFs) are necessary to decodethese signals; RF1 reads UAG and UAA codons, and RF2reads UGA and UAA codons. A third release factor, RF3,is a GTPase dependent on the complex between theribosome and the RFs (Zavialov

et al

., 2001; 2002), whichaccelerates the recycling of RF1 and RF2 (Freistroffer

et al

., 1997). In eukaryotes, a single RF, eRF1, reads allthree stop codons. However, eRF1 in some variant codeorganisms shows more restricted codon recognition(Kervestin

et al

., 2001; Ito

et al

., 2002), in agreement withthe reassignment of one or two stop codons as sensecodons (Lozupone

et al

., 2001). The function of eRF3, theeukaryotic GTPase homologue to RF3, remains unclear(Kisselev and Buckingham, 2000).

The codon specificity of RFs is not well understood atthe molecular level, in contrast to the detailed knowledgeconcerning sense codon reading by tRNAs (Ramakrish-nan, 2002). Functional studies of bacterial RFs have iden-tified one domain as important for peptide release andanother for codon recognition. The first contains the trip-eptide Gly-Gly-Gln (GGQ), the only motif that is perfectlyconserved among RFs from all organisms (Frolova

et al

.,1999). Experimental evidence suggests that GGQ, like theCCA end of tRNA, interacts with the peptidyl transferasecentre (PTC) on the 50S ribosomal subunit, activatinghydrolysis of the ester bond in peptidyl-tRNA (Frolova

et al

., 1999; Seit-Nebi

et al

., 2001; Mora

et al

., 2003;Scarlett

et al

., 2003). The second domain contains a trip-eptide motif that determines the identity of bacterial RFs,as suggested by experiments in which domains wereswapped between RF1 and RF2 (Ito

et al

., 2000). Thetripeptide, Pro-Xxx-Thr (PXT) in the RF1 family and Ser-Pro-Phe (SPF) in the RF2 family, determines whether theRF reads UAA and UAG as RF1 or UAA and UGA as RF2,and the first and third residues of the tripeptide have beenproposed to interact directly with the stop codon on theribosome (Ito

et al

., 2000). How the two factors recognizethe uridine common to the three stop codons is currentlynot understood.

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L. Mora, A. Zavialov, M. Ehrenberg and R. H. Buckingham

© 2003 Blackwell Publishing Ltd,

Molecular Microbiology

,

50

, 1467–1476

It has been suggested that the functional similaritybetween tRNAs and RFs is reflected in a structural mim-icry between these molecules (Cantor, 1979; Nissen

et al

., 1995; Nakamura

et al

., 1996). Therefore, it came asa surprise that the crystal structure of

Escherichia coli

RF2 (Vestergaard

et al

., 2001) was not compatible withthe simultaneous interaction of the SPF region with stopsignal and the interaction of the GGQ region with the PTC.For these interactions to be made simultaneously, theGGQ and SPF regions in RF2 would need to be spacedabout 75 Å apart, the distance between the anticodonregion and the CCA region of a tRNA molecule, whereasin the crystal structure, they were found to be only about23 Å apart (Fig. 1A). Furthermore, attempts to dock theRF2 structure on the 5.5 Å structure of the

Thermus ther-mophilus

ribosome such that the SPF–codon interactionwas made ran into difficulties because of clashes betweenRF2 and rRNA (Vestergaard

et al

., 2001). This apparentcontradiction between functional and structural data ontermination was removed by recent cryoelectron micro-scope (EM) studies of RF2 in complex with the ribosome(Klaholz

et al

., 2003; Rawat

et al

., 2003). These showedthat RF2 has a structure on the ribosome that is verydifferent from the crystal structure and, in particular, thatthe distance between the GGQ and SPF motifs on theribosome may, in fact, be close to the 75 Å necessary tospan the distance between the PTC and decoding centre(Fig. 1C).

In this work, we study the activity of two N-terminallytruncated molecules and four chimeric RF1/RF2 mole-cules in reactions that reflect the interaction with the

codon present in the ribosomal A-site and with the class2 factor RF3. The integrity of the N-terminal domain haslittle importance for RF1 but is required for normal activityof RF2.

In vitro

, the truncated factors are functionallydeficient in their interaction with RF3. Our results lendindependent support to the notion that the SPF and PXTmotifs determine the identity of RFs, and they strengthenthe idea that changes to other parts of the RF moleculeare not required to switch specificity from UAG to UGA. Inthis respect, bacterial RFs appear to differ from eRF1 in‘universal code’ and ‘variant code’ higher organisms (Seit-Nebi

et al

., 2002).

Results

Design of chimeric and truncated RFs

Truncated RFs and chimeric RF1/RF2 molecules weredesigned on the basis of the crystallographic structure of

E. coli

RF2 (Fig. 1). Domain 1 in the crystal structure(Vestergaard

et al

., 2001) is domain A of Ito

et al

. (2000),and the chimeras are referred to here as RF1(A2) andRF2(A1). Truncated factors RF1(

D

A) and RF2(

D

A) wereconstructed by deletion of domain A. Two chimeras wereconstructed by the exchange of domains 1 of RF1 andRF2 with the same choice of domain boundary as thatmade by Ito

et al

. (2000). It should be noted that thesequence of the wild-type RF2 used here corresponds tothat of

E. coli

B and all other sequenced

E. coli

strainsexcept K-12. RF2 from K-12 strains contains Thr at posi-tion 246 instead of Ala, and is several fold less active in

Fig. 1.

A. The three-dimensional crystal structure of

E. coli

RF2, showing functional domains (Vestergaard

et al

., 2001). The SPF tripeptide in

E. coli

RF2 is shown in yellow, within a loop of 13 amino acids (red) in the crystal structure of the molecule. The triple-helical domain 1 is shown in blue/green, where the green residues represent the region absent in

E. coli

RF1. Domain 3 containing the GGQ tripeptide is shown in indigo.B. Primary structure representation of truncated RF1 and RF2 and four chimeric RF1/RF2 molecules in relation to wild-type RF1 (blue) and RF2 (red). The sequences around the junction points in the chimeric molecules are shown, using the colours of the parent sequences.C.

a

-Carbon backbone of a model of RF2 in an unfolded form, based on cryoelectron microscopy (accession number: 1MI6; Rawat

et al

., 2003) and obtained by reorienting domains 1 and 3 of the crystal structure. The same colours are used for domains as in (A), and the central superdomain 2–4 is shown in the same orientation as in (A).

Interactions of truncated and chimeric RFs

1469



,

50

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peptide chain release

in vitro

than

E. coli

B RF2 (Dinçbas-Renqvist

et al

., 2000). The SPF motif is present in RF2within a loop of 13 amino acids that make few H-bondswith other domains of the RF2 molecule, and none thatappears to involve the side-chains of the residues. It there-fore seemed likely that exchanging this 13-amino-acidloop between RF1 and RF2 would not greatly destabilizethe protein structure. This region and the correspondingregion in RF1 were exchanged to create two new chimericRF molecules, RF1(SPF) and RF2(PAT), as shown inFig. 1B.

Specificity of tetrapeptide release by modified RFs

The peptide release activity of the modified RFs wascharacterized for pretermination release complexes, inwhich the ribosome was paused at one of the terminationcodons, UAG, UAA or UGA. The pretermination com-plexes were made as described previously (Freistroffer

et al

., 1997) by the translation

in vitro

of short syntheticmRNAs encoding the tetrapeptide Met–Phe–Thr–Ile,using purified initiation and elongation factors and in theabsence of RFs. The ribosomal complexes, paused at thetermination codon on the mRNA, were separated fromother components of the translation system by gel filtra-tion. Experiments on the release of tetrapeptide by mod-ified and wild-type RFs are summarized in Fig. 2 andTable 1. The truncated molecule RF1(

D

A) and the chimeraRF1(A2) retain the specificity of RF1 with comparableactivity; thus, the factor is active at UAG and UAA codonsbut not at UGA. Both RF2(

D

A) and RF2(A1) have signifi-cantly lower activity than RF2 but retain the same speci-ficity. Their activity is maximal at UGA and significantlyless at UAA.

RF1(SPF) has release activity comparable with the wild-type factors but has acquired the specificity of RF2. Nopeptide release activity was observed with the RF2(PAT)chimera on any of the complexes. These results are con-sistent with the conclusions of Ito

et al

. (2000) that thespecificity of codon recognition is a property of the SPF/

PAT region of

E. coli

RFs. However, they show more clearlythat the specificity of RF1 can be switched to that of RF2without the introduction of amino acids other than SPFand a small number of immediately surrounding residues.

Fig. 2.

Stop codon specificity for tetrapeptide release

in vitro

by trun-cated RF1 and RF2 and RF1/RF2 chimeras. Wild-type or mutant RF molecules were used to release the peptide MFTI from ribosomal release complexes paused at UAG, UAA or UGA codons. The bars show the percentage of tetrapeptide released after 5 s incubation from 2 pmol of ribosomal release complex in the presence of 2 pmol of RF [except chimera RF2 (A1): about 9 pmol incubated for 2 min; and RF2(

D

A): about 4 pmol incubated for 2min].

Table 1.

Summary of functional activities of truncated RF1/2 and chimeric RF1/RF2 molecules.

RF

Release

in vitro

Supports growth StimulatesGDPexchange

Recyclingstimulatedby RF3:GTP

Recyclinginhibitedby RF3UAG UAA UGA RF1 ts

D

RF2

RF1

++ ++

–

++

–

++ ++ ++

RF2 –

++ ++

–

++ ++ ++ ++

RF1(

D

A)

++ ++

–

++

– – – –RF2(

D

A) –

++ ++

–

+

– – –RF1(SPF) –

++ ++

– –

++

a

++

RF2(PAT) – – – – – ND ND NDRF1(A2)

++ ++

–

++

–

+ ++

–RF2(A1) –

+ +

– – ND – ND

a.

Recycles rapidly even in the absence of RF3.ND, not determined.

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L. Mora, A. Zavialov, M. Ehrenberg and R. H. Buckingham



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The modified factors were also tested for their ability tocomplement

in vivo

a thermosensitive mutant of

prfA

(Rydén and Isaksson, 1984) and a chromosomal deletionof

prfB

. Both RF1(

D

A) and RF1(A2), but none of the otherfour factors, allowed growth at 42

∞

C on solid rich mediaof the thermosensitive

prfA

strain, which was restored toabout that of the wild-type strain (results not shown). Ofthe six mutant RFs, only RF2(

D

A) allowed growth of astrain in which

prfB

was deleted from the chromosome,and the rate of growth was slow, about 20% that of thewild-type strain. These results suggest that the specificityof the factors

in vivo

with respect to UAG and UGAcodons, in those cases where the modified factors wereable to sustain growth, was similar to the specificityobserved

in vitro

. Experiments at different temperaturesshowed that the expression of truncated RF1 in strainswith either a wild-type or a mutant chromosomal

prfA

generesulted in cryosensitive growth. RF2(

D

A) expression alsoled to cryosensitive growth of wild-type strains. Thisbehaviour is reminiscent of the cryosensitivity of

prfC

strains (Ehrenberg

et al

., 2000), which may result from thevariation with temperature of RF recycling in the absenceof RF3.

Stimulation of the exchange of GDP bound to RF3 on the ribosome

Recycling of the class 1 factors RF1 and RF2 is stimulatedin

E. coli

by a class 2 RF, the ribosome-dependentGTPase RF3 (Freistroffer

et al

., 1997). When free, thisfactor binds GDP several orders of magnitude morestrongly than GTP, and therefore enters the ribosome inthe GDP rather than the GTP form (Zavialov

et al

., 2001).On the ribosome, GDP must be exchanged for GTP and,owing to the stability of the RF3–GDP interaction, theexchange must be catalysed. Exchange of GDP on RF3with exogenous GDP or GTP requires a ribosome in com-plex with RF1 or RF2. It is therefore feasible to study thespecificity of interaction of class 1 RFs with the A-site-bound codon simply by measuring the stimulation of therate of exchange of GDP bound to RF3. Some mutantclass 1 RFs that have lost the ability to release thenascent peptide chain from the ribosome can neverthe-less interact normally with RF3 and stimulate GDPexchange (Zavialov

et al

., 2002; Mora

et al

., 2003). Thisexperimental approach was used to study the codon-dependent binding of the two truncated RFs and the fourchimeric RF1/RF2 molecules to the ribosome. As shownin Fig. 3, the chimera RF1(SPF) stimulated GDPexchange on RF3 almost as efficiently as RF2 at a UGAcodon, but did not stimulate exchange with UAG at the A-site. This means that RF1(SPF) binds to the ribosome withthe same codon specificity as RF2. As observed previ-ously, the GAQ mutants derived from RF1 and RF2 main-

tain the ability to catalyse GDP exchange like the parentfactors, despite the loss of the ability to catalyse peptiderelease (Zavialov

et al

., 2002), and maintain the codonrecognition specificity of the parent factors. In contrast,neither of the truncated RFs was found to stimulate GDPexchange on RF3 at either UAG or UGA codons (Fig. 3,Table 1). RF1(A2) did not stimulate exchange to an extentcomparable to the wild-type factors or the chimeraRF1(SPF), but some experiments at fivefold higher con-centrations of release complex and RF showed a residualcapacity to promote exchange (results not shown; seebelow).

Recycling of truncated and chimeric RFs

The reduced capacity of both the truncated factors andthe chimeric factors RF1(A2) and RF2(A1) to catalyseGDP exchange on RF3 raised the question as to whetherRF3 is able to promote recycling of these mutant factors.Tetrapeptide release experiments of the type describedabove were therefore performed, but in the presence of alarge excess of release complex over RF. Under theseconditions, the class 1 RF must recycle many times torelease all the peptide from the release complex. Asobserved previously (Freistroffer

et al

., 1997), the rate ofrelease of tetrapeptide by wild-type RF2 (Fig. 4A) andRF1 (not shown) was increased several fold by the addi-tion of RF3 and GTP, as a result of faster recycling of theclass 1 factor. In contrast, little or no increase in recyclingwas seen with either of the truncated factors, consistentwith the idea that RF3 cannot catalyse the recycling ofthese mutant factors (Fig. 4A and C and Table 1). In someexperiments, a significant though variable level of stimu-lation was seen with the truncated factors when RF3 wasadded with GTP; this was unexpected as neither of thesemutants could promote nucleotide exchange on RF3.However, the addition of a low level of GDP (50

m

M, 20-fold lower than the concentration of GTP) to the systemeliminated this stimulation of recycling with the truncatedfactors, whereas it had no effect on the stimulation seenwith the wild-type factors (Fig. 4A and C). We interpret thisto mean that some RF3 can bind to a limited extent to thepost-termination complex directly in the GTP form, or in anucleotide-free form followed by GTP binding to the factor.Because the affinity of RF3 for GDP is about 10

3

higherthan for GTP (Zavialov et al., 2001), the presence of a lowconcentration of GDP means that RF3 will bind directly inthe GDP form.

Experiments with RF1(SPF) showed that the rate ofrecycling of this chimeric factor does not depend on RF3and is as fast as the rate of recycling of wild-type RF2 inthe presence of RF3. The rate of peptide release byRF2(A1) was too low for meaningful conclusions concern-ing recycling of the factor to be drawn. Recycling experi-

Interactions of truncated and chimeric RFs 1471

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 1467–1476

ments with the chimera RF1(A2) were intermediatebetween those with the wild type and the truncated fac-tors. Recycling was clearly stimulated by RF3 and GTP,and the addition of 50 mM GDP did not affect the efficiencyof recycling. Similar results were found with ribosomespaused on UAA codons (Fig. 4D) or at UAG codons (notshown). In spite of the fact that GDP exchange on RF3was not accelerated by RF1(A2) and release complexesunder the same conditions as by the wild-type factors,exchange of GTP for GDP on RF3 evidently takes place,as the presence of GTP was required to stimulate recy-cling of RF1(A2) (Fig. 4D).

Previous experiments led to the conclusion that, in theabsence of guanine nucleotide, RF3 forms a stable com-plex on the ribosome with class 1 factors, preventing recy-cling (Freistroffer et al., 1997; Zavialov et al., 2001). Under

these conditions, the class 1 factor can rapidly releaseone peptide, but it recycles very slowly between ribo-somes. We have tested this behaviour with both truncatedRFs and the RF1(SPF) and RF1(A2) chimeric factors. Asshown in Fig. 5, the recycling of RF1(SPF) was inhibitedby RF3 in the absence of GTP, like the wild-type factor. Incontrast, no stabilization was observed with either trun-cated RF or RF1(A2) (Fig. 5 and Table 1).

Discussion

The experiments described here concern two aspects ofthe function of class 1 RFs: their recognition of stop sig-nals in the ribosomal A-site and how they interact withthe class 2 factor RF3. From the crystal structure of RF2,it was unclear how the factor could bind to the ribosome

Fig. 3. Catalysis of GDP exchange on release complexes by truncated RFs and RF1/RF2 chi-meras. Release complexes (1.5 nM) paused at UAG or UGA stop codons and treated with puromycin were incubated with [3H]-GDP–RF3 (160 nM), class 1 RF (20 nM) and an excess (0.5 mM) of unlabelled GDP.A. mRNA with UGA; no RF (open circles); RF2 (filled squares); RF1(A2) (filled lozenges); RF1(SPF) (open inverted triangles); RF2(DA) (open upright triangles); RF1 (filled upright tri-angles); RF2(GAQ) (open lozenges).B. mRNA with UAG; no RF (open circles); RF1 (filled upright triangles); RF1(A2) (filled lozenges); RF1(DA) (open upright triangles); RF1(SPF) (open inverted triangles); RF1(GAQ) (open squares); RF2 (filled squares). Some results are not shown for reasons of clarity but are summarized in Table 1.

1472 L. Mora, A. Zavialov, M. Ehrenberg and R. H. Buckingham


so that the SPF motif interacted with the stop codon andthe GGQ motif was near the PTC (Vestergaard et al.,2001). Recent studies by cryoelectron microscopy (Kla-holz et al., 2003; Rawat et al., 2003) then showed thatthe conformation of RF2, bound to the ribosome in anactive conformation either before or after peptide release,was incompatible with that found in the crystal. The ribo-somal complexes containing RF2 showed additional elec-tron density extending from the decoding centre on the30S subunit to the PTC on the 50S subunit that wasinterpreted to mean that RF2 is present in an open con-formation on the ribosome in contrast to the compactconformation seen in the crystal structure of Vestergaardet al. (2001). This means that we are still lacking a high-resolution structure of a prokaryotic RF that might helpus to understand in detail the interaction with the ribo-some. Furthermore, it has become clear that there areimportant differences between the ways in which RF1and RF2 bind to the ribosome, as will be discussed fur-ther below. Two ways in which a conformational rear-rangement of the crystal structure of RF2 might beperformed in order to fit the EM models were proposed(Klaholz et al., 2003; Rawat et al., 2003), based on rota-tions of both domain 1 (blue/green in Fig. 1A) anddomain 3 (indigo in Fig. 1A) with respect to the centralsuperdomain 2–4. These would allow the SPF and GGQregions to be simultaneously in close proximity to thedecoding centre and the PTC, respectively, as suggestedby chemical data.

Although the major role in stop codon recognitionplayed by the SPF/PXT region was clearly demonstratedby Ito et al. (2000), it was less clear whether other regionsof class 1 RFs might contribute to the specificity. Such asituation seems to be true in the case of eRF1, wheredifferent parts of domain 1 distant in terms of primarystructure apparently play an important role (Seit-Nebiet al., 2002). The experiments of Ito et al. (2000) wereperformed in a hybrid background, in which some parts ofthe molecule distant from the SPF/PAT tripeptide werederived from RF1 and other parts were derived from RF2.The switch in stop codon recognition that we report herefrom UGA to UAG in RF1(SPF), the maintenance of codonspecificity in the truncated RFs and the absence of anychange in specificity in RF1(A2) or RF2(A1) are in com-plete agreement with the conclusions of Ito et al. (2000;2002) that the transplanted residues are entirely respon-sible for the recognition specificity of the class 1 factor.Here, it is significant that the newly acquired specificity ofRF1(SPF) was obtained by transplanting the SPF loop of13 residues into the otherwise wild-type sequence of RF1,rather than into a complex hybrid sequence. However, thereverse transplant gave rise to an inactive moleculeRF2(PAT), consistent with the conclusion that a hybridbackground may be necessary in order for the switch tobe obtained in both directions (Ito et al., 2000). The rec-ognition specificity of RF1(SPF) was observed both inpeptide release assays and by the capacity of the chimericfactor to catalyse GDP exchange on RF3 at UGA codons

Fig. 4. Catalysis of class 1 RF recycling by RF3–GTP. Release complexes (30 nM) paused at UAG [for RF1(DA)], at UGA [for RF2, RF1(SPF) and RF2(A1)] or at UAA [for RF1(A2)] stop codons were incubated with 1–3 nM class 1 RF in the absence of RF3 (open symbols, solid lines), the presence of 180 nM RF3 and 1 mM GTP (closed symbols, dashed lines), the presence of RF3, 1 mM GTP and 50 mM GDP (half-open symbols, dotted, dashed lines) or 1 mM GDP (in D: crossed lozenges).A. RF2 (1 nM) (squares); RF2(DA) (stars).B. RF2(A1) (circles); RF1(SPF) (inverted triangles).C. RF1(DA) (upright triangles).D. RF1(A2) (lozenges).



but not at UAG codons. The maintenance by the truncatedRFs of the stop codon specificity of the parent moleculesand the results obtained with the chimeras RF1(A2) andRF2(A1) are not consistent with the suggestion (Vester-gaard et al., 2001) that domain 1 plays an important rolein stop codon recognition.

When tested with release complexes appropriate totheir codon recognition specificity, the two truncated RFsand the chimeric factors RF1(A2) and RF2(A1) all lost thecapacity to catalyse GDP exchange on RF3. In the caseof RF2(A1), this might simply be related to a generaldecrease in peptide release activity. RF1(DA) andRF1(A2), on the other hand, maintain a release activity invitro comparable to that of the wild-type factors, consistentwith the fact that they are sufficient for cell growth. Thecapacity of the modified RFs to catalyse GDP exchangeon RF3 is generally correlated with the ability of RF3 to

catalyse the recycling of the factor; thus, neither truncatedfactor was found to recycle any faster between ribosomeswhen RF3–GDP and GTP were present (i.e. in the pres-ence of 1 mM GTP and 50 mM GDP). However, in thepresence of RF3 and GTP, but in the absence of GDP,significant stimulation of recycling of RF1(DA) wasobserved, suggesting that RF3–GTP may be able to pro-mote the dissociation of the truncated factor from the post-termination ribosomal complex as it does with the wildtype factors, in spite of the absence of domain 1.

The chimeric factor RF1(A2) appears to present a caseintermediate between the truncated and wild-type factors.Here, the capacity to promote guanine nucleotideexchange was no longer apparent under the experimentalconditions used, but RF3 could still accelerate recyclingof the factor in the presence of GTP, but not with GDPalone. Nucleotide exchange therefore clearly occurs,although a more detailed study of the kinetic constantsmay be necessary in this case to confirm the validity ofthe mechanism proposed for the wild-type factors. In gen-eral, our experiments clearly point to the importance ofdomain 1 in RF1 and RF2 for the functional, and probablystructural, interaction with RF3 on the ribosome.

A further demonstration of the interaction between class1 and class 2 RFs on the ribosome is the effect on recy-cling of RF1/2 by RF3 in the absence of guanine nucle-otides. Whereas RF1(SPF), like the wild-type factors, waslargely immobilized on the ribosome by RF3 in theabsence of guanine nucleotide after one round of peptiderelease, this was not apparent in the case of RF1(A2) orthe truncated RFs. Taken together, these data show thatthe normal interaction between the class 1 RFs and RF3is strongly perturbed when domain 1 is removed or evenwhen domain 1 in RF1 is replaced by the equivalentdomain from RF2. Like the results on recycling in thepresence of RF3 and GTP, this is also consistent with theorientation of domain 1 proposed by Klaholz et al. (2003)and Rawat et al. (2003) in which the domain approachesthe base of the L7/L12 stalk that contains the GTPase-associated centre, the presumed binding site of theGTPase RF3 on the ribosome. Such an interaction is alsoanalogous to the proposed interaction between the C-terminal domain of eRF1 and eRF3 in the mammaliansystem (Merkulova et al., 1999; Frolova et al., 2000). Fur-thermore, there is a clear parallel between the fact thatthe C-terminal domain of eRF1 can be removed withoutloss of termination activity in vitro (Frolova et al., 2000)and the observation we report here that the N-terminaldomain of the bacterial factors is not essential fortermination.

The results presented here show that RF1 and RF2 arestrikingly different in their capacity to retain activity afterstructural manipulation. Among the truncated and chi-meric RFs we describe, three are sufficiently active to

Fig. 5. Effect of RF3 on recycling of class 1 RFs in the absence of guanine nucleotide. Release complexes (30 nM) paused at UAG [for RF1(A2)], UGA [for RF2 and RF1(SPF)] or UAA [for RF1(DA) and RF2(DA)] stop codons were incubated with class 1 RF in the absence (open symbols) or presence (closed symbols) of RF3:RF2 (1.5 nM) (squares); RF1(SPF) (0.6 nM) (inverted triangles); RF1(A2) (3 nM) (lozenges); RF1(DA) 2 nM (upright triangles); RF2(DA) 1.5 nM (stars).



allow cell growth [although very slowly in the case ofRF2(DA)], two are active in vitro but not in vivo, and oneis totally inactive. This is shown by both experimentsaffecting the codon recognition region in domain 2 and theeffects of deleting or swapping domain 1. Thus, all threemodified forms of RF1, RF1(DA), RF1(A2) and RF1(SPF),were about as active as the wild-type factor in vitro, andthe first two also in vivo. At present, it is not clear whyRF1(SPF), which in vitro displays a stop codon specificityand a peptide release activity comparable to wild-typeRF2, should not be able to restore growth to an RF2-deleted strain; however, this might be explained by a moredetailed kinetic analysis of the activity of the factor. Incontrast to RF1, the corresponding modified forms of RF2were much less active or inactive. Although this does notnecessarily imply that domain 1 plays a different role inRF1 and RF2, these observations may be the reflectionof a distinct difference between the two factors that hasalready been remarked upon by other authors. Despitethe high overall homology between RF1 and RF2, theyappear to bind to distinct although overlapping sites in theL11 region of the ribosome, which is precisely the regionwith which domain 1 of the RFs is thought to interact.Study of the effects on termination, first, of mutationsaffecting helices 43 and 44 of 23S rRNA and, secondly,of deleting the N-terminal domain of L11 have led to thesuggestion that RF2 interacts with the two RNA loops atthe head of these helices, whereas RF1 interacts insteadwith the N-terminal domain of L11 (Tate et al., 1983; Arkovet al., 2000; Van Dyke et al., 2002; Xu et al., 2002; VanDyke and Murgola, 2003). One possible explanation forthe importance of domain 1 in RF2 is that a conforma-tional change in the L11 region is required for the properactivation of the PTC by RF2 before peptide release.

Experimental procedures

Mutant RFs

Construction of truncated RFs and RF1/RF2 chimeras.Wild-type and chimeric RFs used here all contained a C-terminal His6 tag. This has been shown previously not toaffect the activity of RF1 or RF2 in peptide release (Moraet al., 2003). The exchange of domain 1 between RF1 andRF2 to make RF1(A2) and RF2(A1) was based on the intro-duction of an EcoRI site at the domain 1/domain 2 boundaryin prfB and prfA as described by Ito et al. (2000). Both geneswere cloned into plasmid pLV1 (Heurgué-Hamard et al.,2002) giving pLM1(R1) in the case of prfB (Heurgué-Hamardet al., 2002) and an analogous plasmid in the case of prfA.The genes were modified to encode a C-terminal His6 tagon the RF. Truncated factors RF1(DA) and RF2(DA) wereconstructed by replacing the NdeI–EcoRI fragment in themodified prfA and prfB genes by the oligonucleotides5¢-pTATGCAGTTAG-3¢ and 5¢-pAATTCTAACTGCA-3¢. Thisresulted in the sequence E90Q91L92 in RF1 becoming the new

N-terminus fM1Q2L3 and the sequence A111Q112L113 in RF2becoming the new N-terminus fM1Q2L3. Plasmids for theexpression of chimeras RF1(SPF) and RF2(PAT) were con-structed from the parent plasmids by single- or two-steppolymerase chain reaction (PCR) mutagenesis, resulting inthe exchange of regions of 13 residues containing the SPFor PAT regions between RF1 and RF2 (see Fig. 1). Amutagenic oligonucleotide of 101 nucleotides (nt) coveringthis region was used in conjunction with an oligonucleotidein the domain 1 coding region of prfB to amplify the domain2 coding region. The EcoRI–BspEI fragment from this PCRproduct was used to replace the corresponding fragment inplasmid pLM1(R1). An expression vector for RF1(SPF) wasconstructed in an analogous way, except that a second PCRstep was used to extend the first PCR product beyond theBsrGI site in prfA. The EcoRI–BsrGI fragment in prfA wasthen replaced with the corresponding mutated fragment. RFswere purified for in vitro experiments as described previously(Mora et al., 2003).

Activity in vivo of modified RFs

Strain US486 carrying a thermosensitive mutation in prfA(Rydén and Isaksson, 1984), a kind gift from Monica Rydén,was transformed with derivatives of pLV1 carrying the genesfor wild-type, truncated or chimeric RFs (see above) to testfor complementation of the chromosomal mutation. Growthof the transformants at the non-permissive temperature of42∞C was examined on LB plates containing 200 mg ml-1

ampicillin with no IPTG or IPTG at 10-4 or 10-3 M. The prfBgene was deleted from the chromosome in a strain express-ing the gene from a plasmid by homologous recombinationas described by Yu et al. (2000) with a PCR product ampli-fying a tetR–tetA cassette, flanked by 50 nt regions upstreamand downstream of prfB. Recombination resulted in the lysSgene, downstream of prfB, becoming expressed from the tetApromoter. To test whether RF2(DA) or RF2(A1) can supportcell growth in the absence of wild-type RF2, the region con-taining the deleted prfB gene was transduced with phage P1from the prfB-deleted strain to strains expressing the mutantforms of RF2 from pLV1-derived plasmids, selecting for tet-racycline resistance. Viable colonies were obtained withRF2(DA) only when IPTG was present at 10-4 M or higherconcentrations.

Release complexes

Ribosomes and pretermination release complexes were pre-pared as described previously (Freistroffer et al., 1997; Zavi-alov et al., 2002). Polymix buffer, containing 5 mM potassiumphosphate, 5 mM magnesium acetate, 5 mM ammoniumchloride, 95 mM potassium chloride, 0.5 mM calcium chlo-ride, 8 mM putrescine, 1 mM spermidine and 1 mM dithio-threitol (Jelenc and Kurland, 1979), was used in allexperiments. About 80% of the ribosomes were active inpeptide synthesis. Synthetic mRNAs encoding MFTI con-tained an efficient Shine–Dalgarno ribosome binding site, a5 nt spacer region, the coding sequence followed by one ofthe stop codons UAG, UAA or UGA, two sense codons anda poly(A) tail (Zavialov et al., 2002).



Tetrapeptide release in vitro

Release of MFTI tetrapeptide from release complexespaused at UAG, UAA and UGA codons was performedessentially as described by Freistroffer et al. (1997), withminor modifications. The proportion of active RF in purifiedwild type RF1, RF2 and chimera RF1(SPF) was estimatedas described previously by single-round tetrapeptide releaseassays in the presence of excess RF3 with no guanine nucle-otide (Zavialov et al., 2001), and was typically 20–30%. Theactive concentration of mutant RFs deficient in interactionwith RF3 could not be measured as accurately and wasestimated from the two-phase kinetics of peptide release.Quantities or concentrations of RFs given refer to the activemolecules. After incubation for the time indicated in the figurelegends, 50 ml aliquots of the reaction mix were added to anequal volume of 20% formic acid, kept on ice for 15 min andcentrifuged for 30 min at 4∞C at 13 000 r.p.m. in an Eppendorfcentrifuge; 95 ml of the supernatant was taken for scintillationcounting.

Exchange of GDP on RF3 bound to ribosomes

GDP exchange experiments were conducted essentially asdescribed previously (Zavialov et al., 2001; 2002), with minorchanges. [3H]-GDP–RF3 was prepared by incubation for10 min at 37∞C with 8 mM GDP (1200 Ci mol-1), cooled on iceand separated from the bulk of free GDP by gel filtration onSephadex G25 (Pharmacia), pre-equilibrated with polymixbuffer. In a separate reaction mix, 3 nM RC paused at a UAG,UAA or UGA codon was incubated for 20 s at 37∞C with0.2 mM puromycin, 1 mM unlabelled GDP ± class 1 RF (seefigure legends) and mixed with an equal volume of [3H]-GDP–RF3 solution to give a final concentration of 0.16 mM RF3.Aliquots (30 ml) were removed after different incubation times,filtered through nitrocellulose, and the [3H]-GDP retained onthe filter was measured by liquid scintillation counting.

Acknowledgements

This work was supported by the Centre National de laRecherche Scientifique (UPR9073), l’Association pour laRecherche sur le Cancer, the Fondation pour la RechercheMedicale, the Swedish Research Council for EngineeringSciences, the Swedish Natural Science Research Counciland the Swedish Foundation for Strategic Research.

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