Cooperative assembly of proteins in the ribosomal GTPase centre

2396-2403 Nucleic Acids Research, 1995, Vol. 23, No. 13 ©1995 Oxford University Press

Cooperative assembly of proteins in the ribosomalGTPase centre demonstrated by their interactionswith mutant 23S rRNAsGunnar Rosendahl and Stephen Douthwaite*

Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark

Received April 13, 1995; Revised and Accepted May 22, 1995

ABSTRACT

The ribosomal protein L11 binds to the region of 23SrRNA associated with the GTPase-dependent steps ofprotein synthesis. Nucleotides 1054-1107 within thisregion of the Escherlchla coll 23S rRNA gene weremutagenlzed with bisulphite. Twenty point mutations(G—>A and C->T transitions) and numerous multiplemutations were generated. Expression of mutant 23SrRNAs In vivo shows that all the mutations detectablyalter the phenotype, with effects ranging from a slightgrowth rate reduction to lack of viability. Temperaturesensitivity is conferred by 1071G-»A and 1092C->Usubstitutions. These effects are relieved by pointmutations at other sites, Indicating functional inter-connections within the higher order structure of this23S rRNA region. Several mutations prevent directbinding of r-proteln L11 to 23S rRNA In vitro. Thesemutations are mainly in a short irregular stem(1087-1102) and within a hairpin loop (1068-1072),where the protein probably makes nucleotide con-tacts. Some of these mutations also interfere withbinding of the r-protein complex L10.(L12)4 to anadjacent site on the rRNA. When added together torRNA, proteins L10.(L12)4 and L11 bind cooperativelyto overcome the effects of mutations at 1091 and 1099.The proteins also stimulate each others binding torRNA mutated at 1087 or 1092, although in these casesbinding remains clearly substoichlometric. Surpris-ingly, none of the mutations prevents incorporation ofL11 into ribosomes in vivo, indicating that other, as yetunidentified, factors are involved in the cooperativeassembly process.

INTRODUCTION

Understanding the molecular mechanisms involved in ribosomefunction requires an appreciation of the relative contributions ofthe ribosomal protein (r-protein) and rRNA components. Thedirect involvement of rRNA in translation has been firmlyestablished and there is evidence for its interaction with mRNA,tRNA, elongation factors and antibiotics (reviewed in I). Theobservation that rRNA depleted of most r-proteins supports

peptide bond formation (2) suggests that the rRNA also plays acatalytic role. With data accumulating on rRNA function,r-proteins have been conceptually relegated to subsidiary roles,such as protecting rRNA from degradation, stabilizing rRNAconformations or facilitating conformational switches in therRNA. Such functions are, however, undoubtedly essential for theassembly of the ribosome, as well as for its speed and accuracyduring translation.

The 23S rRNA region between nucleotides 1050 and 1110 isassociated with ribosomal processes requiring GTP hydrolysis(3) and Includes the binding site of r-protein Lll (4,5). Thefunction of the region is inhibited by the thiopeptide antibioticsthiostrepton and micrococcin (6,7), the binding of which isdependent on Lll (3). The r-protein complex L10.(L12)4 isimportant for the function of this region and binds adjacently toLll (5,8) in a mutually cooperative manner (9). It is nowdesirable to establish what specific features of the rRNA structureare important for r-protein interaction and to determine theirrelevance for ribosome function within living cells. We reasonedthat mutations in the rRNA might reduce r-protein bindingaffinity to a level where the protein-rRNA interactions could bemore closely followed both in vivo and in vitro.

We have used random chemical mutagenesis to introduce arange of point mutations within a limited region (positions1054—1107) of an Escherichia coli 23S rRNA gene encoded ona plasmid. The physiological effects of the mutations weredetermined by monitoring growth under conditions where cellsare dependent on mutant ribosomes for protein synthesis. Theeffects of the 23S rRNA mutations on the binding of antibioticsand on the in vivo assembly of r-protein LI 1 into ribosomes werefollowed with ribonucleases and chemical probes. Nucleotidesaccessible to these probes were identified by allele-specificprimer extension. Using the same methods, the interactions ofrRNAs with isolated r-proteins were studied in vitro. The dataindicate that the ribosomal GTPase centre is assembled in a highlycooperative manner.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Escherichia coli strain DH1 (10) was used as the plasmid host.Plasmid-transformed cells were grown in LB medium (10) with

* To whom correspondence should be addressed

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Nucleic Acids Research, 1995, Vol. 23, No. 13 2397

ampicillin (Sigma) at 100 |ig/ml or with ampicillin at 25 |ig/mltogether with erythromycin (Sigma) at 30 or 90 |ig/ml. Escherichiacoli strains TGI (10) and CJ236 (11) were used as hosts of M13bacteriophage derivatives and were grown in YT medium (10) at37°C, with added uridine in the case of CJ236 (12). Escherichiacoli strain AM68 lacks r-protein Lll (13).

Construction of templates for bisulphite mutagenesis

The 1 kb Sacl-Sall fragment of the 23S rRNA gene (14) wassub-cloned into Ml3. Single-strand DNA template for site-directed mutagenesis (12) was prepared from strain CJ236. Aunique sequence, to facilitate allele-specific priming, was intro-duced in the 23S rRNA gene around position 1170 (15). Adeletion that removes the 23S rRNA sequence 1054-1107 wasmade using the oligodeoxynucleotide 5'-GGCCGACTCGAGG-CTGTCTGGGCC. This introduces a Xhol restriction endo-nuclease site at the location of the deletion.

DNA heteroduplexes were formed with a single-strandedwindow at the site of the deletions. M13 DNA was prepared fromstrain TGI and 3 |ig replicative form DNA with the abovedeletion was digested with Xhol. The DNA strands wereseparated by heating and heteroduplexes (16) formed on coolingtogether with 2 (xg single-stranded M13mpl8 and mpl9 clonescarrying the undeleted insert. The use of both orientations of thesingle-stranded insert allowed C—»T as well as G-»A transitionsto be obtained.

Mutagenesis and expression of mutants

Bisulphite was used to deaminate cytosines within the single-stranded window of the DNA heteroduplexes (16). The dualmutations 1092C—>U + 1099G—>A and the mutations at position1085 were formed by site-directed mutagenesis (12). Mutationswere identified by dideoxy sequencing. Selected mutant sequenceswere recloned on 0.9 kb Sacl-Sphl fragments into the expressionvector pSTL102 (17) or pLK2058G (18).

Determination of growth rate

Cells harbouring plasmids encoding 23S rRNA were grown at 30,37 and 42 °C on LB agar plates, with and without erythromycin.Colony size was assessed visually and the times required to formdistinct colonies (2 mm in size) were used to estimate the relativegrowth rates of cells (see text to Table 1). Although less precisethan grow rate measurements in liquid culture, we prefer theplating method, as it avoids gross inaccuracies which can arise inliquid culture if a cell undergoes a change (in the plasmid orchromosome) enabling it to outgrow the other cells. On agarplates such changes give rise to isolated, anomalously largecolonies, which can be excluded from the evaluation. PlasmidpKK3535 (14) lacks the 23S rRNA 2058G resistance marker andwas used to determine the efficacy of erythromycin inhibition ofcells with only wild-type ribosomes.

Isolation of ribosomal particles

The fate of mutant rRNAs within growing cells was followed byfractionating ribosomal particles on sucrose gradients (19,20).The relative proportion of mutant rRNA in each ribosomalfraction was analysed by primer extension (15,21). Ribosomesfor biochemical probing experiments were isolated from plasmid-

Q C C A U C A U U - -

3 io«o 4o G Y ' • • • A A 'X A A v ' Y u A n A A G u

A A R C C A Y C A U Y H o A G Y AA B I I I M l C I I I I D

G Y G Q U I I G U A , , Y U CR .,R U U V R « _ A T " A Y

G C2 R - Y

C - Q10»0 T ™" R

R A GA c A

C c c - RQ • U

Figure 1. Secondary structure of the GTPase-associated centre in Escherichiacoli 23S rRNA (5). The stem and loop regions referred to in the text are labelled2-4 and A-D respectively, (a) Transitions from guanine and cytosine that wereisolated as single bisulphite mutations are shown as large letters. Mutagenesis ofthe boxed bases resulted in a large reduction in LI 1 binding; mutagenesis at theother bases caused no or only a slight reduction in L11 binding (Table 2). A1067A1085 and A1095 were altered by site-directed mutagenesis. A1067 and A1095(indicated by arrows) greatly reduce the interaction with thiostrepton andmicrococcin drugs without affecting the binding of LI 1 (23). (b) Phylogeneticconservation of bases in this region. Nucleotides that are conserved in at least 7Cof 72 aligned 23S-hke rRNAs from the Eukarya, Archaea, Bacteria andchloroplasts (31,32) are shown in large letters. Positions conserved as purines oipyrimidines are indicated by R or Y respectively.

transformed cells harvested at an A450 of 0.4. Cells were lysed bysonication and ribosomes were isolated by centrifugation (17).

Binding of antibiotics and probing of complexes

Ribosomes at 0.08 (iM were incubated alone or with a 3-foldmolar excess of thiostrepton or micrococcin antibiotic in 100 pJ50 mM HEPES-KOH, pH 7.6, 10 mM MgCl2, 100 mM KC1, 5mM DTT and 0.2 U RNasin (Promega) for 20 min at 30°C.Ribosomes were probed at 37 °C with dimethylsulphate (DMS)(2 ^1 of a 1:6 dilution in 96% ethanol) for 8 min. Reactions werestopped by phenol/chloroform extraction and rRNA was recoveredby ethanol precipitation (22). Protein-rRNA complexes weremade and probed as described by Egebjerg et al. (5).

Primer extension footprint analysis

The ribosomes in this study are a mixed population containingeither chromosome-encoded, wild-type 23S rRNA or plasmid-


2398 Nucleic Acids Research, 1995, Vol. 23, No. 13

encoded, mutant 23S rRNA. Allele-specific priming was used todifferentiate between the rRNAs. Chromosome-encoded rRNAwas screened using an oligodeoxynucleotide primer complemen-tary to nucleotides 1169-1187 of wild-type 23S rRNA (thewild-type 1170 primer). Plasmid-encoded 23 S rRNA wasscreened using a primer (the mutant 1170 primer) complementaryto the new sequence introduced in the same region (23). The5'- [32P] -end-labelled primers were extended with AMV reversetranscriptase (Life Sciences) as described by Stern et al. (24).

RESULTS

Mutations in the 23S rRNA GTPase domain

The mutagen sodium bisulphite reacted specifically with cyto-sines in the single-stranded region of the gapped heteroduplexes,forming transition mutations within this limited sequence. Thesingle-stranded window, the boundaries of which are defined by

Table 1. Growth rates of cells with pSTL102 plasmids encoding mutant 23S rRNA

the deletion introduced in the 23S rRNA GTPase region,corresponds to the binding sites of r-protein Lll and theantibiotics thiostrepton and micrococcin.

Single point mutations were obtained at 18 of the 23 possibletarget bases in this region (Fig. la). In addition, mutations atpositions 1109 and 1115, just outside the window, were isolatedon separate clones. Individual point mutations and selectedsequences containing multiple mutations were cloned into theexpression vector pSTL102 (Table 1). This plasmid contains theentire rrnB operon with the 2058G marker in 23S rRNA,conferring resistance to erythromycin (25). Sequences withsingle mutations at 1068A, 1072U or 1093A and with somecombinations of multiple mutations could not be cloned intopSTL102. These mutations were inserted into an alternativeexpression vector, plasmid pLK2058G, containing the rrnBoperon with the 205 8G marker under control of the weaker APLpromoter (26).

Mutations in23S rRNAGTPase region

None

1056A

1062A

1064U

1071A

1075U

1076U

1079U

1085G

1O85U

1087A

1091A

1092U

1099A

1100U

1102U

1104U

1107 A

1109U

1115A

1068A+1099A

1071A+1106A

1092U+1099A

1092U+1109U

No erythromycin37°C

1.00

0.88 (± 0.07)

0.92 (± 0.05)

1.00

0.65 (± 0.08)

1.05 (±0.05)

0.95 (± 0.06)

1.00

1.00

1.00

0.67 (± 0.05)

0.97 (± 0.04)

0.62 (± 0.07)

1.00

0.95 (± 0.05)

0.95 (±0.03)

1.00

0.82 (±0.04)

1.02 (±0.02)

0.85 (± 0.07)

0.76 (± 0.03)

0.52 (±0.04)

1.00

0.69 (± 0.05)

Erythromycin (90 mg/1)30°C

1.00

0.88 (± 0.06)

0.73 (± 0.09)

0.79 (± 0.03)

0.55 (± 0.02)

0.93 (± 0.03)

1.00

0.90 (±0.07)

0.81 (±0.10)

0.72 (± 0.02)

0.61 (±0.05)

0.74 (±0.08)

0.66 (±0.10)

0.80 (±0.06)

0.87 (±0.06)

0.77 (±0.08)

0.85 (±0.09)

0.74 (±0.08)

0.94 (±0.06)

0.89 (±0.02)

0.00 (±0.00)

0.58 (±0.06)

0.76 (±0.08)

0.60 (±0.03)

37°C

1.00

0.75 (±0.06)

0.67 (±0.06)

0.82 (± 0.05)

0.49 (±0.02)

0.94 (±0.04)

0.87 (± 0.03)

0.84 (± 0.04)

0.83 (± 0.09)

0.80 (± 0.06)

0.74 (± 0.07)

0.83 (± 0.05)

030 (± 0.04)

0.84 (± 0.05)

0.87 (± 0.06)

0.88 (± 0.04)

0.90 (± 0.05)

0.75 (± 0.03)

1.00

0.84 (± 0.05)

0.12 (±0.12)

0.54 (± 0.06)

0.84 (± 0.06)

0.59 (± 0.05)

42°C

1.00

0.83 (± 0.04)

0.84 (± 0.04)

0.79 (± 0.03)

0.00 (± 0.00)

0.97 (± 0.03)

0.97 (± 0.03)

0.% (± 0.07)

0.87 (± 0.06)

0.84 (± 0.03)

0.78 (± 0.06)

0.80 (± 0.03)

0.00 (± 0.00)

0.83 (± 0.05)

0.83 (± 0.05)

0.95 (± 0.03)

0.91 (±0.04)

0.73 (± 0.04)

0.95 (± 0.05)

0.83 (± 0.03)

0.12 (±0.12)

0.12 (±0.07)

0.97 (± 0.03)

0.33 (± 0.03)

Each of the plasmid-encoded 23S rRNAs has the 2058G mutation, conferring erythromycin resistance, and mutations in the 1170 region for allele-specific priming.The 23S rRNAs additionally have single mutations (or, in four cases, double mutations) in the GTPase region, as indicated. Growth rates of cells were comparedon agar plates without erythromycin and with erythromycin at 30,37 and 42°C and were standardized against the growth of cells bearing a plasmid without mutationsin the 23S rRNA GTPase region. Cells with this latter plasmid formed distinct (2 mm) colonies after an average of 14 h on plates without erythromycin and after18 h (30°C) or 16 h (37 and 42°C) on plates with erythromycin. The grow rates of these cells were normalized to 1. A grow rate of, for example, 0.5 on plates withno erythromycin indicates that these cells took 28 h to form colonies 2 mm in size. The growth rates are means of at least diree experiments; standard errors are shown.



L11-MINUS

rRIBOSOMES-iDMS ' - + + + + •

L11 • • •THIO - - • - - + -

MICR + - - + U G C A

r - 1 1 0 2 U - | - 1 0 6 4 U - i — 1100U-T—1076U - ,- + + + l - + + • I - • + + l - + + + D M S_ _ + _ _ _ + _ _ _ + _ _ _ + _ T H ) 0

+ + _ _ _ + _ _ . + M | C R

1070 * i-t 1067• 1070

1088- -1088

1095- - 1095

Figure 2. Autoradiograms assessing the in vivo assembly of r-protein L11. In the autoradiogram on the left, ribosomes lacking L11 were incubated with combinationsof LI 1 and antibiotics, as indicated above the gel lanes, and were then probed with dimethylsulphate (DMS). Bases protected in 23S rRNA were analysed by reversetranscriptase extension from the wild-type 1170 primer. On the right of the dideoxy sequence lanes (U, G, C, A), ribosomes were isolated from cells with plasmidsencoding the 23S rRNA mutations indicated. Antibiotics were bound and ribosomes were probed with DMS as indicated on the far right. The reactivities of basesin these plasmid-encoded 23S rRNAs were determined by allele-specific extension from the mutant 1170 primer.

Phenotypic effects of the mutations

The growth of cells harbouring mutant rRNA was monitored onagar plates with or without erythromycin. Wild-type ribosomeswere inhibited by 30 mg/1 erythromycin, therefore the drugconcentrations used here allow only ribosomes with the plasmid-encoded 23S rRNA 2058G resistance marker to engage in proteinsynthesis. The phenotypic effects of the mutations in the 23SrRNA GTPase region are therefore more easily detected in thepresence of erythromycin (Table 1). Many point mutations reducethe cell growth rate. Mutants with 1071A and 1092U havemarkedly slower growth at 30 and 37°C and are non-viable at42°C. The 1092U effects are related to disruption of base pairingwith 1099G, as the 1099G->A change relieves the temperature-sensitive phenotype of 1092U. On its own, however, the 1099Amutation has no significant effect on cell growth, even though thiswould also disrupt the 1092-1099 base pair. Temperaturesensitivity in the 1092U mutant is partially relieved by a C—>Usubstitution at position 1109 and the combination of mutation1071A with 1106A enables growth at 42°C.

The single point mutations 1068A, 1072Uand 1093 A are moredeleterious than those listed in Table 1, but, in the absence oferythromycin, cells can tolerate expression of these rRNAs fromthe weaker XPL promoter. However, the cells are non-viable in thepresence of erythromycin at 30 Hg/ml. Analysis of sucrosegradient fractionated ribosomal particles showed that rRNA with1068A, 1072U or 1093A was poorly assembled into 50S subunitsand was only marginally present in 70S ribosome and polysomefractions (data not shown). The results suggest that 23S rRNA

with mutations at 1068,1072 or 1093 functions extremely poorlyand is lethal if expressed in larger amounts from the rrnpromoters. The 1099G—»A substitution eases the effects of1068A and this combination was expressed from the rrnpromoters. Growth on erythromycin could not, however, bereliably sustained (Table 1).

Probing of mutant ribosomes

Proper assembly of r-protein L11 within wild-type ribosomes canbe demonstrated by two characteristic and different footprintprotection patterns on the 23S rRNA. The first of these resultsfrom the protein directly protecting the rRNA from biochemicalprobes (5) and is conspicuously absent in LI 1-minus ribosomes(Fig. 2). The second footprint pattern involves binding of theantibiotic thiostrepton (or micrococcin) to ribosomes (27).Effective binding of these drugs requires the presence of LI 1 (28)and no drug footprint is obtained on LI 1-minus ribosomes (Fig.2). The footprint patterns on mutant ribosomes were studied forany changes that would indicate that the rRNA mutationsinterfere with assembly of r-protein LI 1.

The Nl of unpaired adenines is the position most reactive toDMS detected here by primer extension. Some of the G-»Atransitions therefore directly lead to an increase in modificationat the mutated base. In several cases single G—>A and C—>U pointmutations also caused a more general opening of the rRNAstructure in the ribosomal GTPase centre, particularly aroundposition 1O88A (Fig. 2). However, the accessibility displayed in



the rRNA mutant ribosomes is lower than that observed inLll-minus ribosomes (29; Fig. 2).

The presence of LI 1 in these mutant ribosomes is most clearlydemonstrated by the r-protein-dependent binding of drugs. Inribosomes containing Lll the accessibility to DMS of position1067A is reduced by thiostrepton, but is enhanced by micro-

coccin, while both drugs protect position 1095A (27). Thesefootprint patterns were found in all ribosomal particles with singleor double bisulphite mutations (Table 2), indicating that proteinLll had been assembled. Reduced drug binding was evident,however, when the rRNA structure had been disrupted by fourbisulphite mutations.

Table 2. Binding of r-proteins to 23S rRNAs with mutations in the GTPase region

Mutations in 23SrRNA GTPase region

None

1056A

1062A

1064U

1068 A

1071A

1O72U

1075U

1076U

1079U

1085G

1O85U

1087A

1091A

1092U

1093 A

1099A

1100U

1102U

1104U

1107A

1109U

1115A

1O64U+1O75U

1068A+1099A

1071A+1106A

1092U+1099A

1092U+1109U

1106A+1071A

1052U+1072U+1092U+1100U

1064U+1072U+1075U+1102U

1092U+1102U+! 104U+1109U

LI 1 bindingto naked23S rRNA

+

+

-

+

(+)

-

-

+

+

+

-

-

(+)

(+)

-

-

(+)

(+)

(+)

(+)

(+)

+

+

-

L10.(L12)4

binding tonaked 23S rRNA

+

+

(+)

(+)

+

+

Cooperativebinding ofLll +L10.(L12)4

+

+

(+)

+

(+)

+

(+)

In vivoassembly of Lllinto nbosomes

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

(+)

(+)

Binding complexes between r-proteins and naked 23S rRNAs were formed in vitro with a 3:1 molar excess of protein (5). The degree of binding of L10.(L 12)4 andLI 1 to naked rRNA was estimated from the intensities of the RNase V| footprint patterns (Table 3): +, stoichiometric binding; (+), much reduced binding (<50%);- , no binding (<5%). A blank space indicates not determined. The in vivo assembly of LI 1 inribosomes was estimated by the anubiotic footprint patterns widi DMS(Fig. 2). It is not clear whether the last two sets of multiple mutations reduce drug binding directly or whether they do so indirecdy by interferring with LI 1 assembly.



In vitro interactions of r-proteins with mutant rRNAs

Many nucleotides in the naked 23S rRNA structure are accessibleto RNase V] (30). Within the region studied here the same siteswere accessible in wild-type and mutant rRNAs, suggesting thatthe rRNAs fold into the same overall conformation. The bindingof r-proteins LI 1 and L10.(L12)4 to naked 23S rRNA can bedemonstrated in vitro by RNase V] footprinting (Table 3). Pointmutations at any one of 14 different sites within the LI 1 bindingsite reduce binding of the protein to naked 23S rRNA (Table 2).The chromosome-encoded, wild-type rRNA, which is present ineach of the mutant rRNA preparations, was analysed as aninternal control and showed unaltered Lll binding.

Table 3. The reactivities to RNase V) of nucleotides within the GTPaseregion of naked, wild-type 23S rRNA (5; Fig. 3)

Nucleotide No protein +L11 L10.(L12)4LI0.(L12)4

1031G

1039A

1040A

1044C

1060U

1061U

1079C

1080A

1081U

1090A

109IG

1118C

Nucleotide reactivities were graded by visual estimation from gel autoradio-grams. I M i, very reactive; +, weakly reactive; - , unreactive. Stoichiometncbinding of the r-proteins L10.(L12)4 and LI 1 changes the reactivities as shown.A blank space indicates no change.

We investigated the interaction of L10.(L12)4 with several ofthe mutant 23S rRNAs that had shown defective Lll binding(Table 2). The single mutations 1087A, 1091 A, 1092U and1099A also reduced binding of L10.(L 12)4 to the rRNA (Fig. 3).Lll and L10.(L12)4 mutually stimulate binding when addedtogether to the 1091Aor 1099A mutant rRNAs. Taking 1099AinFigure 3 as an example, addition of LI 1 alone slightly enhancesRNase V i cutting at position 1031 and marginally protects around1060 and 1080, all these effects being indicative of weak binding.Addition of L10.(L12)4 alone slightly enhances cutting at 1031and 1039, but there is no protection at 1044 or enhancement at1118, also indicative of impaired binding. When the sameamounts of protein are added in conjunction, positions 1031,1039 and 1118 are markedly enhanced and 1044, 1060 and 1080are completely protected, as would be expected for stoichiometricbinding of Lll andL10.(L12)4. Binding also improved when theproteins were added together to the 1087A or 1092U mutantrRNAs, but the lack of complete nucleotide protection shows thatbinding remained sub-stoichiometric (Fig. 3).

DISCUSSION

Many of the point mutations in the GTPase region of 23S rRNAinterfere with protein synthesis. This becomes evident whengrowing cells are forced (by addition of erythromycin) to relyexclusively on mutant ribosomes. Considering the conservation ofthis rRNA region in Bacteria, Archeae, chloroplasts and Eukarya(31,32), it is to be expected that some structural changes causeadverse effects. The secondary structure model (Fig. lb) shows thepositions of invariant and semi-invariant bases. Point mutations atsome of these positions have drastic effects on cell growth, as wasobserved for the mutations at 1068, 1072 and 1093, which greatlyreduce the ability of 23S rRNA to function in protein synthesis.However, the adverse effects on the phenotype do not alwayscorrelate with the degree of base conservation, as can be seen forthe invariant positions 1079, 1085, 1100 and 1104, wheremutations have little effect on growth.

As there has been strong evolutionary selective pressure againstchanges at these latter positions, it can be envisaged thatmutations here might be more deleterious under other growthconditions. Such conditional effects are illustrated by the 1071Aand 1092U mutations, which are tolerated at 30 and 37 °C, but arelethal at higher temperatures. Mutation at position 1099 relievesthe temperature-sensitive phenotype of the 1092 mutation,presumably by restoring base pairing (Fig. 1). Less obvious ishow the temperature-sensitive phenotypes are partially relievedby secondary mutations (1071A by 1106A and 1092U by 1109U)at sites which appear to be independent in the secondary structuremodel (Fig. 1). The functional interconnection between thesenucleotides could be mediated either by the tertiary folding of thisrRNA region or through the r-proteins attached there.

The effects of the rRNA mutations on the in vivo assembly ofr-protein Lll were determined by biochemical probing togetherwith allele-specific primer extension. In ribosomes from cells inwhich LI 1 has been genetically deleted (13), the redundant proteinbinding site on the rRNA is exposed and reactive to chemicalmodification (29). Some of the rRNA point mutations also increasethe accessibility of nucleotides in this region, although not to adegree that suggested that Lll is absent (Fig. 2). The antibioticprotection data unambiguously verified that none of the bisulphite-induced single or double mutations prevents in vivo assembly ofLll into ribosomes. Cells with the 1071A and 1092U mutationsdisplay a temperature-sensitive phenotype similar to theLI 1-minus strain. At 37°C, LI 1 is present in ribosomes with theserRNA mutations (Table 2). However, it is possible that someLll-dependent function is impaired at higher temperatures.

Mutations affect the interactions of the isolated 23S rRNA andLll components in vitro much more than is apparent fromribosome assembly in vivo. Binding of L11 to naked 23S rRNA isgreatly reduced by each of the bisulphite mutations in loop B andin helix 4 and also at positions 1062,1093,1104 and 1107. Severalof these bases are probably sites of protein-rRNA contact, anddamage selection (33) and mutagenesis studies (34) on RNAfragments containing the protein binding site have also implicatedbase interactions in LI 1 binding. It is unlikely, however, that all ofthese positions are involved in protein contact Some of themutations, particular at nucleotides involved in base pairing, couldreduce protein binding indirectly by perturbing the rRNA structure.Consistent with this, E.coli Lll binds to phylogenetically diverse23S rRNAs which have large sequence differences, but whichpresumably have similar secondary and tertiary structure (35,36).



WT—P-1092UI -1099A-J— 1091A

lL11L10.(L12)4

1031-

1039-1044- .

1087A—.—1075U—,—1104U

+ + + +UGCA + + + + + ++ + + +UGCA>~- 1031

• - - 1039

~ - 1044

1060-

1080-

- 1060

~ 1080

- 1090

1090-

1118-- 1118

Figure 3. In vitro binding of r-proteins to 23S rRNAs. Naked 23S rRNA was incubated with r-proteins L10.(L12)4 and/or LI 1 and was probed with RNase Vj, asindicated at the top left of the figure. The sites of the RNase cuts in chromosome-encoded 23S rRNA were analysed by extension from the wild-type 1170 primer (lanesWT); the plasmid-encoded 23S rRNAs containing the point mutations indicated above the lanes were extended from the mutant 1170 primer. The figure is a compositeof two autoradiograms that were run under near identical conditions.

Base 1092C does, however, appears to be a site of protein contact.Lll binding remains impaired even after the secondary structureof 1092U has been restored with 1099A.

Several mutations which do not affect Ll l binding areclustered in helix 3. Hydroxyl radical probing of the L11-23SrRNA complex indicated that the protein makes extensiveinteractions with the rRNA backbone in helix 3, but not in helix4 (29). Taken together, these sets of data suggest that Ll lrecognizes and binds to 23S rRNA mainly through contact withthe backbone of helix 3 and with bases in loop B and helix 4.

Footprinting experiments have shown that the L10.(L12)4complex and LI 1 bind to adjacent overlapping sites on 23S rRNA(5), probably in a cooperative manner (9). Single rRNAmutations, including those at positions 1087, 1091, 1092 and1099, reduce protein binding to a level where mutual stimulationof Ll l and L10.(L12>4 interaction can be clearly followed byfootprinting. Figure 3 illustrates how addition of the proteinstogether greatly improves their binding to the 1091A and 1099ArRNAs. Cooperative protein interaction also clearly takes placeon the 1087A and 1092U mutant rRNAs, but the footprint effectsare less intense, reflecting a lower stoichiometry of proteinbinding. In vivo, however, protein Lll assembles with each ofthese mutant rRNAs, indicating that other factors are involved insubunit formation.

As the r-proteins are properly assembled in vivo, the questionremains as to how the rRNA mutations in Table 1 impair cellgrowth. The ribosomal GTPase region performs an array offunctions involving transient interactions with elongation(37,38), initiation and release factors. It has already beensuggested that these interactions necessitate conformationalswitches in the region, and that the antibiotics thiostrepton andmicrococcin inhibit such switches by interacting with loops B andD of the rRNA (23,39) to lock the region in one conformation(3,5,27). The impaired interactions of mutant rRNAs withr-proteins in vitro (Table 2) could be an indication of reducedability of this region to interact with factors and/or undergoconformational switches in vivo. The function of the regionrequires coordinated interactions between the r-proteinsL 1O.(L 12)4 and L11 and the rRNA. This is stressed by the data onthe overlapping binding sites of Lll and L10.(L12)4, theircooperative effects and the interplay of rRNA mutations in theL10.(L12)4 and LI 1 sites in reversion from temperature sensitivity.

ACKNOWLEDGEMENTS

Anders Liljas and Roger Garrett are thanked for gifts of r-proteinsLll and L10.(L12)4. The work was supported by grants from the



Carlsberg Foundation and The Danish Natural Sciences Council.GR received a PhD grant from Odense University.

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Cooperative assembly of proteins in the ribosomal GTPase centre