Site-specific mutation of the conserved m62 A m62 A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase

Biochimica et Biophysica A cta, 1050 (1990) 18- 26

18

Elsevier BBAEXP 92150

Site-specific mutation of the conserved m6A m6A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase Philip R. Cunningham 1, Carl J. Weitzmann i, Kelvin Nurse 1, Remco Masurel Peter H. Van Knippenberg 2 and James Ofengand 1

2,

1 Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ (U.S.A.) and 2 Department of Biochemistry, Gorlaeus Laboratory, Leiden University, Leiden (The Netherlands)

Key words: 16S RNA mutagenesis; 16S RNA methylation; Ribosome function; ksgA methyltransferase

In vitro synthesis of mutant 1KS RNA and reconstitution with ribosomal proteins into a mutant 308 ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could he assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity. The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50%. Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function. The mutant ribosomes could also he methylated by the ksgA methyltransferase to 70-120% of the expected amount. Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.

Introduction One of the striking hallmarks of the ribosome, whether from organelles or cytoplasm, from lower prokaryotes or higher eukaryotes, is the conservation of its fundamental construction parameters. Nowhere is this more evident than in the two large R N A molecules which constitute up to two-thirds of the mass of the ribosome. These RNAs contain both conserved sequence segments, and extensive conserved secondary structural elements [1] as well as conserved elements of tertiary structure [2]. Numerous nucleotide modifications, mainly but not exclusively methylations, are also found in ribosomal RNAs [1]. Some are highly conserved while others appear to be characteristic of only a sub-set of species. The two adjacent N6,N6-dimethyl adenosines (m62A) residues in the loop of a small highly conserved stem-loop structure near the 3' end of small subunit ribosomal RNAs are perhaps the best example of a highly conserved base methylation in ribosomal RNA [1,3]. Moreover, the A residues themselves, posi-

tions 1518 and 1519 in the E. coli numbering system [4], are universally conserved [1,3]. Previous studies have shown that lack of dimethylation as it occurs in kasugamycin-resistant mutants of E. coli [5], has only minor effects in vivo and in vitro (reviewed in Ref. 6). The recent development of an in vitro method for the generation of site-specific mutant ribosomes [7] has now made it possible to study the effect of more drastic alterations at these positions on protein synthesis in vitro. Fig. 1 indicates the region under study with respect to the overall 16S RNA structure, and also shows the sites of the mutations studied in this work. Fig. 2 highlights interesting features of the 3' minor domain where these mutations were made. The in vitro mutagenesis system has also allowed us to probe the effect of mutation of one A residue on the methylation of the other. This latter question is important in view of a sequential mechanism that has been proposed for the methylation reaction [13].

Materials and Methods

General techniques

Abbreviations: HSW, high salt wash; DTT, dithiothreitol; EF-Tu, elongation factor Tu; IF, initiation factor. Correspondence: J. Ofengand, Roche Institute of Molecular Biology, Nutley, NJ 07110, U.S.A.

All proeexlures, materials and buffers not otherwise specified were as described [14,16]. Oiigonucleofide synthesis was performed in the trityl-on mode. After manual deprotection, purification was by solid phase

0167-4781/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

19

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Fig. l. Location of the mutated nueleotides. The sequence and secondary structure of Escherichia coli 16S RNA is according to Ref. 8. The sites of methylation are not shown. The inset shows the mutated positions described in this work in bold face type.

extraction using oligonucleotide purification cartridges (Applied B i o s y s t e m s ) following the manufacturer's protocol. For annealing, the purified oligomers were di-

luted to 2 0 / ~ M in annealing buffer [7] c o m b i n e d in a 1 : 1 ratio, heated to 70 o C, and allowed to cool to r o o m temperature over a 30 m i n period. The annealed

20

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Fig. 2. The 3' terminal region of 16S rRNA of E. coli with the eonseawedelements[3] highlighted(boxes). Other featuresincludethe site of ero~linking with the antieodon of bound tRNA [9], the cleavagesite after treatmentof ribosomesby colieinE3 [10]or cloacin DF13 [11], and the nueleotides that interact with mRNA during translationinitiation.

oligomers were diluted to 0.5 #M in water prior to ligation. Small amounts of plasmid DNA for sequencing were isolated from 1.5 ml of an overnight culture grown in TB [17] containing 100 # g / m l ampicinin essentially by the method of Ish-Horowicz and Burke [18] except that the sample was not phenol extracted. Instead, the isopropanol-precipitated material was redissolved in 100 ~1 of TE containing 100 /xg/ml DNAse-free RNAase A (Sigma) and incubated at 37°C for 20 rain. Purified plasmid was obtained by the addition of 0.25 ml of saturated NaI and adsorption of the DNA to 10 #1 of a Geneclean suspension (Bio 101, La JoUa, CA) according to the manufacturer's protocol. After washing according to the protocol, the DNA was eluted with water. Small scale preparations of plasmid DNA for retransformation were prepared by the method of Bimboim and Doly [19] except that a single colony of the original transformant was used instead of an overnight culture. CsCl-purified plasmid DNA was prepared according to Maniatis et al. [20], except that the overnight cultures were grown in 100 ml of TB containing 100 # g / m l ampicillin and were not amplified. The vector for cassette mutagenesis was pWK1 which had been double-digested with BstEII and Bsu36I. It was isolated by loading 1 #g into each well of a 14 cm 1% agarose gel prepared with TAE buffer [20] containing 0.5 # g / m l ethidium bromide. After electrophoresis for 1-2 h at 100 V, the vector band was cut out of the gel, dissolved at 50 °C in 2.5 vol of saturated NaI and the DNA recovered by adsorption to 5 #1 of a Geneclean suspension. Elution was with water as described above. Construction of mutants by cassette mutagenesis Two complementary oligodeoxynueleotides corresponding to the pWK1 sequence between and including

the BstEII and Bsu36I sites were synthesized, except that random nucleotide substitutions were programmed at the position corresponding to residue 1518 of the 16S RNA in one oligomer, and at position 1519 in the other oligomer. The two oligomers were annealed as described above. The heteroduplex (0.5 pmol) was added to 0.1 pmol of agarose-purified vector DNA which had been double-digested with BstEII and Bsu36I, and the mixture ligated for 4 h at room temperature as described previously [7]. The resulting mixture was used to transform competent DH5 cells. Ampicillin-resistant transformants were selected on LB agar containing 100 /~g/ml ampicillin. 24 colonies were chosen. Plasmid DNA was prepared from each and used to retransform DH5 cells. The retransformants were selected on LB agar containing 100 # g / m l ampicillin. One colony from each plate was picked for further study. Plasmid DNA was prepared from each of the chosen colonies and the mutations were identified by sequencing through both ligation junctions. Using this procedure, six mutations at two loci were generated with a single heteroduplex. Plasmid sequencing 10 #g (3.5 pmol) of plasmid DNA was denatured in 0.2 M NaOH and 0.2 mM EDTA for 5 min at room temperature, and the DNA was precipitated with 0.3 M NaOAc/2 vol EtOH. The pellet was washed with 70% ethanol, dried in vacuo and stored at - 2 0 ° C . For sequencing, the denatured DNA was resuspended in 10 #1 of 20 mM Tris-HC1 (pH 7.5), 10 mM MgC12, 25 mM NaC1 and 0.2 #M primer, incubated for 15 min at 37 °C and allowed to cool to room temperature. The primer used corresponded to residues 2003-2025 of pWKI [14] numbered according to the 16S RNA sequence. Dideoxy sequencing reactions were performed using modified T7 DNA polymerase (Sequenase, U.S. Biochemicals) according to the manufacturer's protocol. Transcription Plasmid (15 #g) was linearized with Bsu36I and transcribed in a 1 ml reaction containing 40 mM TrisHC1 (pH 8.0), 8 mM Mg(OAc)2, 25 mM NaC1, 2 mM spermidine, 10 mM dithiothreitol, 1000 units RNAsin (Promega Biotech), 2.5 mM each of ATP, CTP, UTP and GTP, 5 units inorganic pyrophosphatase (Sigma) and 1000 units T7 RNA polymerase (New England Biolabs) at 37 ° C for 5 h. 12 units of RNase-free DNase (Cooper Biomedical) were added and the incubation was continued for an additional 15 rain. The mixture was extracted with phenol/chloroform and the high molecular weight RNA was purified by Sephacryl $200 column chromatography in 50 mM Hepes (pH 7.3), 100 mM NaCI and 10 mM EDTA. The transcripts were recovered by ethanol precipitation and dialyzed versus 5 mM KOAc (pH 5), 1 mM Mg(OAc)2 before storage at - 7 0 ° C. RNA transcripts were routinely checked for

21 sequence in the mutated region and for integrity by electrophoresis after glyoxal-Me2SO denaturation [15].

Total 30S ribosomal proteins (TP30) Two procedures were used with essentially equivalent results. The acetic acid procedure was modified from Nierhaus and Dohme [21] as described previously [7] except that exposure to acetic acid was reduced from 45 min to 15 min. The LiCl-urea procedure was modified from Traub et al. [22]. 30S ribosomal subunits dissolved in RB buffer [7] at 300-400 A260 units/ml were mixed with an equal volume of 8 M urea (Ultrapure, IBI) - 4 M LiC1 (pre-treated with activated charcoal and filtered) at 0 °C for 15 h. After removal of the RNA by centrifugation, the protein supematant was dialyzed versus Rec-20 buffer (five changes of 1 h each). Determination of protein concentration and storage conditions were as described in Ref. 7. 30S subunit reconstitution Reconstitution conditions were as previously described (Legend to Fig. 3, Ref. 15), except that following purification of the reconstituted particles by sucrose gradient centrifugation, the 30S subunits were washed and concentrated in RD buffer by ultrafiltration using 10K Omega cells (Pharmacia). Ribosomes were activated [23] by heating for 30 min at 40 °C in RD buffer, and stored in small aliquots at -140 ° C. HPLC analysis of ribosomal proteins The ribosomal protein content of reconstituted particles was analyzed as described in Ref. 15, except that values were normalized to those of synthetic reconstituted 30S rather than isolated 30S. Materials mRNA for the I site and fMet Val dipeptide assays was made in vitro from plasmid pT7/T3-18 (BRL) which was modified by replacement of the sequence between the EcoRI and HindlII sites with a synthetic deoxyoligomer pair designed to generate the RNA sequence G G G A G A C C G G / A A U U C A A A A U UAAGGAGGAUCCCAU[AUGGUU]UUUAUUACC A G U A / . . . where the first two codons of the peptide, fMet and Val, are enclosed in brackets and the postulated Shine-Dalgarno nucleotides are underlined. The solidi denote the segment inserted between the EcoRI and HindlII sites. The plasmid, denoted pND1, was linearized by cutting with AoalI (New England Biolabs) and was transcribed, and the RNA was isolated as described above for the mutant rRNAs. The molar concentration of mRNA was calculated assuming 40 /~g/A260 unit, an average 340 daltons/base, and a length of 767 residues from the start of transcription to the Avail site. The yield was 640 mol RNA per mol DNA template. A single band co-migrating with the 0.78 kb

marker (0.16-1.77 kb RNA Ladder, BRL) was obtained upon glyoxal-Me2SO denaturing gel electrophoresis. Ribosomal high salt wash (HSW), the source of initiation factors, was prepared by grinding 50 g of frozen, washed, 3 / 4 log phase E. coli MRE600 cells (Grain Processing) with 100 g of alumina in a mortar at 4 ° C. 1 mg of DNase (RNase-free, Cooper Biomedical) was added and the mixture was incubated at 4 ° C for 10 rain. The mixture was suspended in 50 ml of buffer Bill (10 mM Tris (pH 8.2, 60 mM KOAc, 14 mM Mg(OAc)2, 1 mM DTT) and centrifuged, the pellet washed with 25 ml of buffer Bill, and the combined supernatants were cen.trifuged at 105000×g for 4 h at 4°C. The supernatant and fluffy layer were removed, and the ribosome pellet was suspended in 30 ml Bill containing 1 M NH4C1 with stirring for 15 h at 4 ° C. After clarification by centrifugation at 10000 × g for 15 min, the ribosomes were removed at 105 000 × g for 4 h. The HSW supernatant was precipitated with 85% ammonium sulfate, the pellet was dissolved in 5 rnl of buffer Bill and dialyzed vs. the same buffer with two changes for 16 h. The precipitate which formed was removed by centrifugation and the supernatant was stored in small aliquots at - 140 ° C. 30S and 50S subunits were prepared as described previously [7] except that recovery from the sucrose gradient was as described above for 30S subunit reconstitution. The subunits were stored in RD buffer at -140°C. ksgA Methyltransferase was prepared as described previously [24]. The enzyme preparation displayed one major band at approx. 30 kDa on a polyacrylamide gel.

Functional assays P site binding of tRNA. Reaction mixtures contained 50 mM Hepes (pH 7.5), 50 mM NH4C1, 15 mM Mg(OAc)2, 5 mM Dq'T, 20 # g / m l poly(U2,G), 100 nM 50S subunits and 67 nM 30S subunits in a 25/~1 volume. After 10 min of incubation at 37°C, 115 nM Ac[3H]Val-tRNA was added and incubation continued for 20 min. Duplicate samples were analyzed as described [7]. Values for the complete system minus 30S subunits were used as blanks. Blanks with poly(U2,G) omitted were approximately the same as those lacking 30S subunits. A site binding. This assay was performed in duplicate using 40 nM 30S equivalents by the procedure previously described [16]. Binding in the absence of elongation factor Tu (EF-Tu) was less than 10% of that in its presence for all the mutants studied. Blank reactions lacked 30S subunits. Phe, Val Copeptide synthesis. This assay was performed as described [16] except that the concentration of poly(U2,G) was 4 #g/ml. This concentration was still saturating.

22

30S initiation complex formation (1 site). Reaction mixtures contained 55 mM Hepes (pH 7.5), 135 mM NHaC1 , 16-18 mM Mg(OAc)2 (except where indicated otherwise), 5 mM DTT, 1.4 mM GTP, 200 nM mRNA, 1.2 or 3.6 mg/ml HSW, 150-200 nM f[3H]Met-tRNA and 70-140 nM 30S subunits in a volume of 25 or 50 #1. After 20 min at 37 ° C, the mixtures were filtered as described above for P site binding. Values from reactions lacking 30S subunits were subtracted• Binding in the absence of HSW was less than 3% of that in its presence for all of the mutants studied, over the entire range of Mg 2+ concentrations used. fMet-Val dipeptide synthesis• The reaction mixture in 35 #1 contained 35 mM Hepes (pH 7.5), 34 mM NHnOAc , 22 mM NH4C1, 41 mM KOAc, 6 mM KC1, 7.5 mM Mg(OAc)2, 1.2 mM spermidine, 3 mM DTT, 27 mM potassium phosphoenolpyruvate, 11 # g / m l pyruvate kinase, 3 mM ATP, 0.3 mM GTP, 110 units/ml RNasin (Promega), 4.3% poly(ethylene glycol) 8000, 0.5 mg/ml HSW, 700 nM EF-Tu, 230-460 nM mRNA, 300-600 nM unlabeled fMet-tRNA, 200 nM [3H]ValtRNA, 2-20 nM 30S subunits depending on their activity, and a 1.5 to 6-fold excess of 50S subunits over 30S. After incubation for 90 min at 37 ° C, the amount of

fMet-Val formed was assayed as described [16] and was proportional to the amount of 30S subunits added. Values from reactions without 30S subunits were subtracted. Direct comparison of this mRNA-dependent assay with the coupled transcription-translation assay described previously [16] using synthetic wild-type and four mutant 30S preparations showed that both assays were equivalent.

Methylation Methylation reaction mixtures contained 100 mM Tris (pH 7.5), 53 mM NH4C1, 2 mM Mg(OAc)z, 5 mM /3-mercaptoethanol, 7 #M S-[3H]adenosyl methionine, purified and characterized as in Ref. 25. 17 nM 30S ribosomal subunits, and purified ksgA methyltransferase. Incubation was at 37 ° C. Subtracted blank values were from complete reactions lacking 30S subunits. Results

Mutant ribosomes Mutant synthetic 16S RNA was prepared following the described modifications to our previous procedures. Since the RNA synthesis is performed in vitro, all

50 NAT

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FRACTIONNUMBER Fig. 3. Reconstitution of natural and mutant ribosomes. Samples were prepared and analyzed as described in Materials and Methods. 1 ml fractions were collected. Filled circles, A2~o; open circles, [32P]pCp-30S added as an internal marker. Filled ovals indicate the fractions pooled for functional analyses. Each point was calculated by dividing the actual value for A26o or 32p by the total recovered from each gradient. Thus the areas under all of the peaks are equal. The coefficient of reconstitution (Cr) shown in each panel was obtained by taking the ratio of the shaded area to the total A26o area. Integration of the shaded area was performed numerically by s u m m i n g the lesser of the A~x,o or 32p percentages for each fraction.

23 TABLE I Protein content of mutant reconstituted 30S subunits

Analyses were described in Materials and Methods. Values are expressed as protein/RNA mole ratios, normalized [15] to reconstituted synthetic 30S. Values < 0.7 or > 1.3 are underlined. Protein $2 $3 $4 $5 $6 $7 $8 $9 S10 Sll S12 S13 S14 S15 S16 S17 S18 S19 $20 $21

Mutant ribosome C1518

G1518

U1518 C1519 G1519

U1519

0.9 0.9 1.1 1.1 1.0 1.1 1.0 1.0 1.4 1.6 1.0 1.5 1.1 1.0 b 1.1 1.2 1.0 b 1.0 1.1 0.8

1.1 1.0 1.1 1.2 1.0 1.0 1.1 1.1 1.5 1.3 1.1 1.3 1.1 1.1 b 1.2 1.3 1.1 b 1.1 1.1 0.9

0.6 0.8 1.0 1.0 a 0.9 1.0 0.9 1.0 a 1.1 1.4 1.0 1.5 1.1 1.1 b 1.1 1.2 1.1 b 1.0 1.2 0.8

0.7 0.9 0.9 1.1 1.0 1.0 1.1 1.0 1.4 1.0 1.1 0.7 1.1 1.1 1.1 1.0 0.9 1.0 1.0 0.9

0.5 0.7 0.8 1.0 0.9 0.8 1.0 0.9 1.4 0.9 1.1 0.7 0.8 0.8 1.0 0.9 0.8 0.9 0.9 0.7

0.5 0.7 0.8 0.9 0.8 0.7 0.9 0.8 1.1 0.8 1.0 0.6 0.8 0.8 0.8 0.9 0.7 0.9 0.8 0.7

a.b For pairs of proteins not sufficiently resolved, the measured protein content was divided equally between both [15].

m u t a n t s give e q u a l l y g o o d yields [16]. R e c o n s t i t u t i o n was p e r f o r m e d following the s a m e p r o t o c o l used with o t h e r m u t a n t s [15], a n d as shown in Fig. 3, was e q u a l l y successful. A s n o t e d before, r e c o n s t i t u t i o n of n a t u r a l R N A , that is R N A isolated f r o m 30S subunits, gave particles ( N A T ) i n d i s t i n g u i s h a b l e f r o m i s o l a t e d 30S s u b u n i t s , whereas all synthetic particles ( S Y N ) assemb l e d less well. W e have a t t e m p t e d to q u a n t i t a t e the degree of ' c o r r e c t ' a s s e m b l y b y c a l c u l a t i n g a coefficient o f r e c o n s t i t u t i o n (Cr), as d e s c r i b e d in the legend to Fig. 3. O n this basis, as well as b y visual i n s p e c t i o n of Fig. 3, there does n o t a p p e a r to b e a n y significant difference a m o n g the m u t a n t s . T h e w i l d - t y p e synthetic r i b o s o m e s do, however, a p p e a r to a s s e m b l e slightly m o r e efficiently t h a n the m u t a n t s . T h e sucrose g r a d i e n t analysis also served as a p r e p a r a t i v e p r o c e d u r e , since o n l y those fractions s e d i m e n t i n g with the m a r k e r 30S envelope, i n d i c a t e d b y e n l a r g e d symbols, were selected for functional analysis. T h e r i b o s o m a l p r o t e i n c o n t e n t of each of the reconstituted m u t a n t r i b o s o m e s is given in T a b l e I. Overall, all of the p r o t e i n s were p r e s e n t in a p p r o x i m a t e l y unit stoichiometry. T h e S1 c o n t e n t c a n n o t be m e a s u r e d b y this technique, a n d it is n o t k n o w n to w h a t extent it is p r e s e n t in either o u r r e c o n s t i t u t e d or i s o l a t e d 30S subunits. In a few cases, the s t o i c h i o m e t r y of a given p r o t e i n varies f r o m u n i t y b y m o r e t h a n 30%. These are

u n d e r l i n e d in T a b l e I. H o w e v e r , since there is n o correl a t i o n with a d e c r e a s e (or increase) in a given p r o t e i n a n d the f u n c t i o n a l activity o f the particles (as shown b e l o w in T a b l e II), the o b s e r v e d v a r i a t i o n is p r o b a b l y n o t significant. Function in protein synthesis T h e f u n c t i o n a l activity of the six m u t a n t s s t u d i e d in this w o r k is s h o w n in T a b l e II. T h e P site, A site, (Phe,Val), a n d f M e t - V a l asays have b e e n used previously to a n a l y z e o t h e r m u t a n t s [16]. T h e b i n d i n g of f M e t - t R N A to 30S r i b o s o m e s (I site) has b e e n a d d e d to the repertoire, since it is the o n l y p h y s i o l o g i c a l l y relev a n t assay w h i c h does n o t require a s s o c i a t i o n with the 50S subunit, a n d in a d d i t i o n m e a s u r e s the first step of p r o t e i n synthesis initiation. T h e M g 2÷ a n d i n i t i a t i o n factor d e p e n d e n c e of f M e t - t R N A b i n d i n g to 30S subunits in this a s s a y is shown in Fig. 4. A s p e r f o r m e d , the a m o u n t of b i n d i n g was strictly d e p e n d e n t on a c r u d e i n i t i a t i o n f a c t o r p r e p a r a t i o n , a n d h a d a d e f i n e d M g 2÷ d e p e n d e n c e . It was also strictly p r o p o r t i o n a l to the a m o u n t of 30S s u b u n i t s a d d e d ( n o t shown). A s was the case with the assays d e s c r i b e d p r e v i o u s l y [16], n a t u r a l r e c o n s t i t u t e d 30S s u b u n i t s were a l m o s t indistinguishable f r o m i s o l a t e d 30S, while the s y n t h e t i c 30S p a r t i c l e s were a b o u t half as active. The fMet-Ser dipeptide-coupled transcription-transl a t i o n assay d e s c r i b e d p r e v i o u s l y [16] was m o d i f i e d b y r e p l a c e m e n t o f the t r a n s c r i p t i o n c o m p o n e n t s with an m R N A m a d e in vitro f r o m a suitable p l a s m i d . This m R N A c o n t a i n e d a s t r o n g S h i n e - D a l g a r n o sequence s u i t a b l y l o c a t e d 5 ' to the i n i t i a t i o n c o d o n A U G , which was followed b y a G U U codon. This allowed use of V a l - t R N A , one o f the m o s t stable A A - t R N A s , as the a c c e p t o r a m i n o a c i d in the d i p e p t i d e assay. T h e m R N A d e p e n d e n c e is s h o w n in Fig. 5A, which also illustrates the r e q u i r e m e n t for 30S subunits. U n d e r these c o n d i -

TABLE II Functional effects of site-specific nucleotide substitutions

Assays were performed as described in Materials and Methods. The values are expressed as percent of a synthetic wild-type sequence reconstituted at the same time with the same TP30 preparation. Each mutant ribosome was reconstituted twice with a separate preparation of RNA, and the average value was rounded to the nearest 5% which is judged to be the level of accuracy of our measurements. Mutant

tRNA binding

Peptide synthesis

P site

A site

I site

fMet-Val

Phe, Val

C1518 G1518 U1518

70 65 60

55 55 45

100 80 95

45 35 30

120 95 110

C1519 G1519 U1519

65 60 70

55 55 65

60 75 70

50 55 70

85 75 75

24

0.6 O O

0.5

A 0.4 E

V

0.3

11.1 _m i 6

, •

10

14

-" "18

i

--'LLI"

22

26

rnM ~ + +

Fig. 4. Mg2+ and factor-dependent fMet-tRNA binding to 30S ribosomes. The assay was as described in Materials and Methods at the Mg2+ concentrations indicated. Open symbols, plus initiation factors (HSW); filled symbols, without HSW. o, isolated 30S; zx, 30S reconstituted from isolated 16S RNA; v, 30S reconstituted from 16S RNA transcribed in vitro.

tions, the a m o u n t of p r o d u c t formed was proportional to the a m o u n t of 30S added (not shown). As indicated in Fig. 5B, the M g 2+ concentration o p t i m u m was different for synthetic 30S and isolated 30S subtmits. The Mg 2+ concentration chosen for use in the assays was 7.5 raM, since the important c o m p a r i s o n to be m a d e in this work was between wild-type and m u t a n t synthetic ribosomes.

Recognition of the mutant ribosomes by the ksgA methyltransferase It has been suggested that there is an ordered sequence to methylation of A1518 and A1519 by the ksgA methyltransferase, A1519 being methylated first [13]. The synthetic ribosomes described above were used to test this proposal, since having been m a d e in vitro they lacked all R N A methylations [15]. If there was a strict order to the methylation reaction, mutation at 1519

TABLE III

rB

'A

The results of our analyses are summarized in Table II where all activity data are expressed as percentages of the control wild-type sequence ribosomes. Despite the universal conservation of the two A residues, their change to a n o t h e r base did not block any in vitro activity completely. There were, however, certain trends which could be discerned. The P, A, and I site binding values for all six m u t a n t s ranged between 55 and 70%, except for the I site values for the 1518 mutants which were, as a class, higher. Initiation-independent peptide b o n d formation (Phe, Val), like I site binding, was also slightly higher for the 1518 mutants c o m p a r e d to the 1519 series. The strongest effect was on formation of the first peptide b o n d (fMet-Val) in the 1518 series where G or U were only 1 / 3 as active as the control c o m p a r e d to almost full activity for the first step in initiation (I site). Even C1518 was only half as active in dipeptide formation as in I site complex formation. By contrast, the activity o f the 1519 series was only slightly reduced in dipeptide formation c o m p a r e d to the I site assay. This was partially because the I site values for the 1519 series were lower than for the 1518 mutants.

Extent of methylation of synthetic wild-typeand mutant 30S ribosomes by purified m~A raethyltransferase Assays were performed as described in Materials and Methods. For series 154 and 155, values at 30, 60 and 90 rain incubation times were averaged. For series 169, values at 5, 10, 20, 40 and 60 rain were averaged. All of the time points used were at the kinetic plateau. Percent is found x 100/expected. Normalized percent is the ratio of mutant to wild-type percent x 100.

6

c~ 5 E ~-

4

E

2

d

i

Reconsti- Nudeotide Methylation tution position series 1518 1519 found expected L

"

::~.,~, | I"1, 0.2

0.4

f

0.6

pM mRNA

d

0 .8

I

I

6

I

7

!

8

154

A C G U

A A A A

moles CH 3/mole 30S 2.73 4.0 1.63 2.0 1.32 2.0 1.26 2.0

68 82 66 63

100 121 97 93

155

A A A

A G U

3.73 1.33 1.38

4.0 2.0 2.0

93 67 69

100 72 74

169

A' A

A C

2.10 1.01

4.0 2.0

53 51

100 98

I

g

mM MO++

Fig. 5. Mg2÷ and mRNA dependence of fMet-Val dipeptide formation. Panel A: mRNA dependence. 30S subunits were at a concentration of 15 nM, MB2+ was 6.5 raM. Filled circles, 30S r~constituted from 16S RNA transcribed in vitro plus isolated 50S subunits; open circles, isolated 50S subunits alone. Panel B: Mg2+ dependence. Open circles, isolated 30S plus 50S subunits; filled circles, 30S reconstituted from 16S RNA transcribed in vitro plus isolated 50S subunits.

Per- Normalized cent percent

25 should block methylation of A1518, but mutation at 1518 should not affect methylation of A1519. If both A residues were required, no methylation should occur. Preliminary experiments carried out by D. Nrgre and two of us (P.v.K. and J.O.), showed that either crude or purified ksgA methyltransferase could methylate both ksgA 30S subunits (30S subunits from the ksgA mutant which lacks methylation at both A1518 and A1519) and synthetic 30S at approximately equal rates. Thus, synthetic wild-type sequence ribosomes were methylatable in vitro. However, there was considerable variability in the stoichiometry obtained. Some preparations of the ksgA 30S and synthetic 30S were only methylatable to as little as half the expected amount, even with excess enzyme, while others were fully methylatable. Examples of this variability for which the reasons are not known can also be seen in Table III. The methylation results summarized in Table III show that none of the mutant ribosomes block methylation of the unmodified A residue. When the normalized percent of the expected amount was examined, 70-120% of the maximal possible incorporation of methyl groups was obtained. In each case, the nature of the bound methyl was examined by HPLC analysis after digestion of the 16S RNA to nucleosides [21], and only m62A was found. Specifically, m6A was completely absent.

Discussion Mutation of either A1518 or A1519 to C, G or U had little effect on the ability of the mutant RNA to reconstitute a 30S ribosome containing a full complement of ribosomal proteins. In particular, the $21 content of all of the mutants was close to normal. $21 is known to interact with this loop region of 16S RNA, since in excess it inhibits methylation of A1518 and A1519 [24]. Nevertheless, it is clear from this work that neither A1518 nor A1519 constitute a major element of recognition for this protein. Mutation of the A1518 and A1519 residues had little effect on ribosome function. Most activities were only partly inhibited, 50% or less, although initiation-dependent formation of the first peptide bond was two thirds inhibited in the 1518 mutants. This latter result is in agreement with previous studies which showed that lack of methylation of the two A residues resulted in a requirement for more IF3 to attain maximal formation of initiation complexes [26], and with the finding that IF3 binds to this region of 16S RNA [27]. In another study, IF1 formation was also associated with the 3' end of 16S RNA [28]. Sequences may be conserved because they optimize function rather than being required for function. This seems to be the case with the A1518 and A1519 substitutions. While there was no strong decrease of any

activity, most were partially decreased relative to the wild-type sequence. It seems reasonable to propose that the presence of the two A residues, along with some of the other nearby conserved elements, serves, to optimize the 30S structure for its protein synthesis function. Alternatively, since in vivo conditions are normally suboptimal compared to in vitro reactions which are typically adjusted to give maximal effects, the mutant ribosomes may be relatively less active than their wildtype counterparts when functioning in vivo. This remains to be shown. Another factor to consider is that with the exception of the Phe, Val assay, none of the other functional tests used in this work measure rates of reaction. Conceivably, the effect of the mutations on the rate of tRNA binding could be greater than on its extent. In vivo, this could become important and might account for the universal conservation of the A1518 A1519 sequence. The ability of the ksgA methyltransferase to methylate either of the A residues in the absence of the other one was unexpected since there is evidence [13] that methylation is an ordered process. This could mean that m62A1519 formation was strictly required before methylation of A1518 could occur, or only that the enzyme had a kinetic preference for one A residue over the other. In light of the results obtained above, it appears that the latter is the more correct interpretation. Our results also demonstrate that both A residues are not needed for recognition by the methyltransferase, that none of the other bases in place of A are inhibitory and that prior methylation at G1516 is not required for ksgA methyltransferase activity.

Acknowledgements We thank Didier Nrgre for the construction and preparation of plasmid pND1 used for preparation of the synthetic mRNA for the fMet-Val assay. Part of this work was supported by a Collaborative Research Grant (0228/89) of the NATO Scientific Affairs Division to P.H.v.K. and J.O.

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