Nucleic Acids Research, Vol. 18, No. 22 6537

Codon recognition in polypeptide chain termination:site directed crosslinking of termination codon to Escherichia coli release factor 2 Warren Tate*, Barbara Greuer' and Richard Brimacombe1 Department of Biochemistry, University of Otago, Dunedin, New Zealand and 'Max Planck Institute for Molecular Genetics, Berlin, FRG Received August 15, 1990; Revised and Accepted October 19, 1990

ABSTRACT An RNA synthesized in vitro was positioned on the Escherichia coil ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site. Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor. An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition. An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination. The 4-thioU crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified. RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found. If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted. These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon. INTRODUCTION The termination event of protein synthesis occurs when the incoming codon to the ribosomal A site is, in most genetic systems, one of the three triplets, UAA, UAG, UGA. A polypeptide chain release factor then binds to the ribosome and mediates the release of the completed polypeptide from the peptidyl tRNA in the ribosomal P site (1). The physical relationship between release factor and the codon, and the

ribosomal position of the codon during the recognition event has not been established unequivocally. Indeed two diverse models could explain the existing information: (i) that the release factor is in close physical contact with the codon itself at the ribosomal decoding site, and (ii) that the termination codon interacts first with the ribosomal rRNA, thereby allowing the release factor to bind. In the second case there would be no necessity to have close physical contact between the factor and codon, but the binding site for the factor could be created through a conformational change in the ribosome, allowing it to interact with the peptidyl transferase centre and mediate release of the polypeptide. The ribosomal binding site of the Escherichia coli RF-2 is at the ribosomal subunit interface with a domain on the small subunit, but this domain is on the opposite side to that of the decoding site, as determined from immuno electron microscopy. The RF-2 molecule penetrated into the cavern of the large subunit but it was not possible from this study to determine how far the factor extended across to the decoding site (2). Both regions of the 30S subunit have been implicated in the termination mechanism. Spectinomycin, which binds to helix 34 of the l6SrRNA (4) overlapping the factor binding domain, affects RF-2 reactions specifically (5). Hygromycin and neomycin bind near the decoding site of the small subunit (6), quite distant from helix 34, and these antibiotics affect all termination reactions with both bacterial release factors RF-I and RF-2 (5). RF-2 crosslinked to L7/L12, L2 and LII in a functional termination complex, a result consistent with the position determined by immunoelectron microscopy, but also weak crosslinks were obtained with S6,S17,and S18, suggesting a domain of the factor might be in contact with the decoding site (6). However, radiolabelled termination codon crosslinked to many ribosomal products after UV irradiation, including L7/L12, L2 and L1O, quite distant from the decoding site, and to S6 and S18 near the decoding site (7), suggesting the codon might occupy two sites during the course of the termination event. The termination codon can be recognised outside of the decoding site when a ribosome has both P and A sites occupied by codons and

Baylor College of Medicine, 1 Baylor Plaza, Houston, + Present address: Institute of Molecular Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA

*

To whom correspondence should be addressed at Institute of Molecular Genetics,

Texas 77030, USA

6538 Nucleic Acids Research, Vol. 18, No. 22 cognate tRNAs (8,9),

or can be displaced from the A site and still be recognised; if there is direct RF/codon recognition then the release factor must have flexibility in its codon recognition domain without losing specificity (8). An Escherichia coli mutant with specific UGA suppressor activity has a base deletion in the l6SrRNA (1054-in helix 34), and it was noted that there are complementary tandem UCA triplets between nucleotides 1199-1204 in the middle of the same base-paired stem as the deleted nucleotide (10). This region is placed far from the decoding site in the current models for the tertiary structure of the 16SrRNA (4,11) but within the RF-2 binding domain, and would imply a significant shift in the termination codon position from the A site following the last translocation event if an interaction occurs between nucleotides 1199-1204 and UGA. In the current study we have set out to answer two questions: (i) whether RF-2 is in close physical contact with the termination codon, by using site directed crosslinking from the stop codon to the factor, and (ii) whether the codon position changes significantly after the RF-2 is bound into a termination complex, by comparing the crosslinking patterns to ribosomal proteins and rRNA before and after the RF-2 is added to the complex.

MATERIALS AND METHODS In vitro synthesis of [32p] labelled RNA The oligodeoxynucleotide synthesis and mRNA transcription have been described (12 ), except that the T7 polymerase was purified from an overproducing strain of bacteria Where 4-thioU was required in the RNA, UTP was replaced by the 4-thio-UTP in the transcription. The 4-thio-UTP was prepared from 4-thio-UDP (Sigma) as described (12). The mRNA was radiolabelled by using [a&32P]-ATP in the transcription. The resulting RNA product was purified on a 15% polyacrylamide gel containing 7M urea and 0.1 % SDS. The band was located by autoradiography, excised from the gel, extracted with phenol /SDS buffer and collected by ethanol precipitation. The yield (nmol) was calculated from the specific activity of the ATP in the transcription, and generally resulted in 5-10 molecules of mRNA per molecule of template. .

Isolation of [35S]labelled RF-2 An overproducing strain of RF-2 was a kind gift of Dr Uli Goeringer, with permission from Dr Rob Weiss, namely Escherichia coli SU1675 F'IQ with plasmids, pPylO25, and pKB4. Cells were grown in M9 medium containing thiamineHCI (20Ag/ml), amino acids without Met or Cys (201tg/mi), supplements of Trp and Tyr each at 1OAg/ml, 1mM MgSO4, 0.4% glucose, 0. 1mM CaCl2 and the antibiotics ampicillin (100lg/ml) and spectinomycin (50 mg/ml). After reaching early log phase (A6wom of 0.4) IPTG (1.5 mM) was added to induce expression of RF-2 (under control of a Tac promoter and repressed by the lac IQ gene product). After 1 h [35S] Met (500itci) was added and incubation continued for 3.5 h. The proteins from a cell extract were fractionated on a DEAE Sephadex A50 column (10cmx2cm) with a gradient of KCI (SOmM-SOOmM in 200ml). The [35S] labelled RF-2 eluted as the major radiolabelled peak towards the end of the gradient and its identity was confirmed both on an SDS gel and by its ability to bind to ribosomes in response to termination codon.The latter half of the peak gave a homogeneous RF-2 preparation.

A [32P]mRNA.[35S]RF-2. tRNA.ribosome complex The labelled mRNA was added to 150 -200pmol 70S ribosomes (preincubated at 370 for 15min) at a 1.5-2.0 fold excess, tRNAala or thr at a 5 fold excess, and labelled RF-2 at 1 -2 fold excess in 600tl of a buffer containing, 20mM Tris-HCl pH 7.4, lOOmM NH4Cl, 20mM MgCl2, with or without 6% (v/v) ethanol. Addition of the latter stabilised the RF-2 in the complex and prevented partial dissociation during gradient centrifugation. The ribosomes, highly active in poly Phe synthesis, were a kind gift of Dr K.H Nierhaus. The complex was incubated for 30 min at 37°. Where appropriate these complexes were sedimented through a sucrose gradient (10-30%) in the same buffer at 19,000rpm for 16h , 4°. Fractions were collected and analysed for radioactivity. Where [32P] and [35S] were counted together conditions were set so that the spillover from the [32p] into the [35S] channel was - 10%.

Site-Directed Crosslinking from the 4-thio-U in the mRNA The ribosomal complexes (6001l) at 40 were dispensed into plastic dishes (1mm thickness of solution) and irradiated without cooling for 15 minutes with ultraviolet light (>300nm) at a distance of 5cm. The light source was a bank of Philips 15 watt lamps (26cm long, with four of them mounted 5cm apart), catalogue number T129D 16/09N, wavelength range 320-400nm, with a maximum about 350nm, and it provided complete coverage of the samples during irradiation. The samples were returned to 40 following the irradiation, athough there was no evidence of evaporation or excessive warming during the procedure.The complexes were then precipitated with ethanol for buffer exchange. They were then centrifuged through a sucrose gradient under conditions to separate the 50S and 30S ribosomal subunits, and the appropriate fractions containing radiolabelled mRNA crosslinked to ribosomal components were collected, precipitated with ethanol for exchange into an SDS/EDTA buffer as described (13) so that the 16SrRNA and ribosomal protein fractions could be isolated on a subsequent sucrose gradient.

Analysis of the crosslinked complexes of mRNA and other components (a) [35S]RF-2. [32P]mRNA crosslinks The fraction from the top of the subunit gradient containing putative RF-2.mRNA crosslinks was precipitated with ethanol and redissolved in a small volume of 50mM Tris-HCl, 50mM KCI, 1mM DTT. Samples were fractionated on a 12% SDS polyacrylamide gel, the gel cut into 5mm slices and, after digestion with a Soluene/PPO solution, the radioactivity in each slice determined. There was 3-4% spillover from the 32P label (the mRNA label) into the 35S ( the RF-2 label), and only where the mRNA label was in gross excess, in the 3-4 fractions at the bottom of the gel, was assessment of 35S not possible. -

-

(b) Identification of rRNA nucleotides involved in crosslinks The mRNA.rRNA crosslinked fraction was concentrated by ethanol precipitation, and redissolved in water. Aliquots were first hybridised with pairs of deoxyribo-oligonucleotides, complementary to the rRNA at positions 100-200 nucleotides apart, and digested with RNase H as in (14), except that the hybridisation was carried out at 550 C for 10 min., after which the mixture was made 1mM in Mg(OAc)2, 0. 1mM in DTT, the enzyme added and incubation continued for a further 30 mn. at 550 C. The digestion was stopped by the addition of 1/ 10 vol.

Nucleic Acids Research, Vol. 18, No. 22 6539 of 1% SDS, 20mM EDTA-KOH, pH 7.0. This mixture was applied to a 5% acrylamide gel for analysis (14 ).This method allows a preliminary localization of the crosslink site on the rRNA. For final localization of the crosslink site a reverse transcriptase analysis was made. For this purpose the mRNA.rRNA crosslinked fractions were further purified by affinity chromatography on oligo dT cellulose (13 ) but at 40 instead of room temperature. The rRNA, not bound to the column in the buffer (5OOmM NaCl, lOmM Tris-HCl pH 7.8, 1mM EDTA) and hence not containing a polyA sequence from the mRNA, was used as control in each, case as described below. The bound mRNA.rRNA, after extensive washing with application buffer was eluted in the same buffer without NaCl. About 50% of the radioactivity was recovered in the purified polyA+ fraction, with small amounts being lost at each wash. After ethanol precipitation the crosslinked samples were taken up in water. To analyse the position of the crosslink by reverse transcriptase analysis, an oligonucleotide primer was chosen on the basis of the RNase H digestion result (above). The deoxyribo-oligonucleotide, in this case an oligonucleotide complementary to positions 1492-1502 in the 16SrRNA was first hybridised at 700 at 10 fold excess over substrate (0. 1 -0.55Ag) to both the control and crosslinked samples in a buffer containing 50mM K-Hepes pH 7.0, 5mM K borate, and lOOmM KCI. After slow cooling to 42°C dCTP, dGTP, and dTTP were added at -50AM together with 6itCi d[32P]ATP (>400 Ci/mmol) and reverse transcriptase (final concentration 0.4U/4u) (Boehringer) in a reaction buffer consisting of 50mM Tris-HCl, pH 8.5, 50mM KCI, lOmM MgCl2, 10mM DTT. Sequencing reactions were carried out with the same control rRNA samples and template oligonucleotide, but dNTP/ddNTP mixtures were used in this case. Reactions were incubated for 45 min. at 42°C, and then stopped with an equal vol. of 0.2 M NaOH, 25mM EDTA, heated for 5min. at 90°C. After neutralisation with 0.2M HCI the products were precipitated in 2 vol. ethanol at -20°C,together with 2.Ot41 glycogen (10mg/ml), and were taken up in a small volume for application to an 8% sequencing gel.

Dalgarno sequence) (ii) to radiolabel the mRNA with[32P]ATP and, (iii) for subsequent purification of the mRNA.rRNA crosslinks away from the non-crosslinked rRNA on oligo dT cellulose. Recognition of the stop codon by release factor A reliable direct assay for the stable binding of RF to the ribosome is not available. To date this has been measured indirectly from the specific incorporation of radiolabelled ternination codon into a RF.ribosome complex (15). Multiple samples can be analysed rapidly with this assay, but it has limitations because the complex is relatively unstable. Designed mRNAs as illustrated in Fig 1 have the potential to provide a much more stable environment for the RF on the ribosome, and thereby provide a direct assay for binding RF to the ribosome in stiochiometric amounts. This was an important GGAGAAAAAAG ACC GCG UAA GAAAAAAACAAAAAAA thr

ala

stop

polyA tail

Figure 1. The mRNA designed for site-directed crosslinking

*

4-thio

(c) Identification of ribosomal proteins Fractions from the SDS-sucrose gradients containing ribosomal proteins crosslinked to mRNA were pooled and then divided into aliquots with 300-1000 [32P]cpm. Those proteins containing crosslinked mRNA and hence radioactivity were identified using a specific assay with agarose-linked antibodies against each ribosomal protein as described (12).

RESULTS AND DISCUSSION Design of the mRNA A mRNA was designed to be positioned on the Escherichia coli ribosome with a tRNA at the P site, and with a termination codon in the A site as shown in Fig 1. Highly tRNA-dependent binding of such RNAs to ribosomes with tRNAala at a GCG codon had been demonstrated previously (12). A Thr codon was placed upstream from the Ala codon so that the mRNA could also be bound at the P site with tRNAthir, and then the termination codon would be displaced by one codon from the A site. The mRNA contained two stretches of A's which served several purposes (i) to position the Ala codon away from the G rich T7 promoter sequence (which could have mimicked a Shine-

Figure 2. The binding of RF-2 to a ribosomal complex containing designed mRNA. A. Binding of [32P]labelled mRNA to 70S ribosomes. Labelled mRNA at a 1.5 fold molar excess over ribosomes was bound with tRNAala (-o-o-) or without tRNA (-) and sedimented through a sucrose gradient (5-20%w/v). Sedimentation is from right to left. B-F. Binding of [35S] labelled RF-2 to 70S ribosomes. B. In the absence of mRNA. C. In the presence of mRNA. Labelled mRNA was at a 2-fold molar excess, and RF-2 equimolar to ribosomes. The complex contained tRNA,a". D. With mRNA containing 4-thio-UAA in place of UAA. An azidophenacyl derivative was also attached to the 4-thio-U; the binding of the RF-2 in this case is shown by the dotted line.E.With mRNA containing UAC in place of UAA. F. With mRNA and tRNA'hr in place of tRNAala

6540 Nucleic Acids Research, Vol. 18, No. 22 prerequisite in the current study if site-directed crosslinking from the mRNA to the RF were to be possible. The [32p] labelled mRNA (Fig 1) bound to ribosomes stoichiometrically (when the mRNA was present at 2 fold excess the binding was 50% of the input RNA, at 1.5 excess it was 75 %) (Fig 2 panel A). Stable binding was tRNA-dependent; some mRNA attached to the ribosome without the tRNA but it dissociated as the complex was centrifuged through the gradient (broken line -panel A). This complex provided a substrate for a specific assay for stable binding of release factor by the following criteria: (i) the [35S]labelled RF-2 did not bind to the ribosome in the absence of the mRNA (panel B) (ii) when the UAA codon in the mRNA was in the ribosomal A site there was almost quantitative stable binding of the RF-2, added at an equimolar ratio to the ribosomes (panel C) (iii) when the UAA in the mRNA was changed to UAC the RF-2 binding was almost completely abolished (panel E) (iv) if the mRNA was also bound to the ribosome with tRNAfhr so that the termination codon was displaced from the A site, the binding of the [35S]RF-2 to the ribosomes was considerably reduced (panel F), although greater than that seen with the 'UAC' mRNA. This latter condition was of interest because earlier studies with short oligonucleotides had suggested that the RF could recognise the codon when it was displaced from the A site ( 8,9 ). If some of the tRNAthr binds into the E site, thereby still positioning the stop codon at the A site then this would account for RF-2 binding. Current studies however, indicate the E site is filled by tRNA only after the P site is fully occupied (16). Since we wished to carry out site-directed crosslinking from the termination codon the 'U' in the termination codon in the mRNA was replaced by 4-thio-U. This change did not affect the ability of the RF-2 to recognise the stop codon and bind stoichiometrically (panel D). In contrast, when an azido phenacyl derivative was introduced at that site (12) binding of the factor was less stable (dotted line panel D). This study not only established a direct assay for stable binding of release factor to the ribosome but also demonstrated that sitedirected crosslinking from a 4-thio-U residue within the termination codon to the RF should be possible, if the two were in close contact during the codon recognition event.

Site-directed crosslinking Site-directed crosslinking was carried out from the 4-thio-U of the termination codon in the mRNA under several conditions: when the mRNA was bound with tRNA ala in the absence of RF, or the presence of RF, and when the mRNA was bound with tRNAthr without RF. These experiments aimed at addressing two questions, firstly whether the RF and termination codon were in close physical contact and second whether the crosslinking patterns to the ribosomal components changed when the RF was in the ribosomal complex. The tRNAthr was used as a control to determine whether altered crosslinking patterns could be detected when the termination codon within the mRNA was deliberately shifted by one codon with respect to the ribosome. The 70S ribosomal complexes of mRNA.tRNA. ribosome. (+ /-RF-2) were irradiated with light of 320 -350nm to induce crosslinking from the 4-thio-U, and the complexes were resolved on sucrose gradients under dissociating conditions to separate the ribosomal subunits. Under these conditions mRNA not crosslinked to the ribosome will remain at the top of the

gradient, along with the free RF-2, and tRNA, but this fraction would also include any mRNA. RF or mRNA. tRNA crosslinked complexes. Complexes arising from the crosslinking of the mRNA to a ribosomal component will appear as radioactivity associated with one of the subunits within the gradient. As shown in Fig. 3 (panels A and B) a significant proportion of the mRNA was crosslinked with the 30S subunit but not with the 50S subunit. RF-2 reproducibly decreased the percentage of the mRNA sedimenting with the 30S subunit from -50 to - 15-25% of the bound mRNA (panelA-note in this Fig the uncrosslinked peak also includes the unbound mRNA). When tRNAthr was used in place of tRNAala to fix the mRNA onto the ribosome, so that the 4-thio-U was displaced by the length of 1 codon, - 80% of the bound mRNA crosslinked to the subunit, 10 fold higher than that crosslinked in the absence of tRNA (panel B). The 30S fractions were then dissociated in SDS/EDTA and the rRNA and ribosomal protein fractions resolved on a sucrose gradient (panels C and D). In the absence of RF-2 the proportion of radiolabelled mRNA in each fraction was similar (- 50%), while in the presence of RF that crosslinked to the 16SrRNA decreased to -30% (panel C). In the case of crosslinked complexes from the mRNA bound with tRNAthr the majority of the crosslinked mRNA was in the 16SrRNA fraction ( - 80%). In the absence of tRNA, where the absolute amounts are several fold lower, most was at the top of the gradient in the protein fraction, although this will include a 'background' of uncrosslinked mRNA which had carried through to the 30S subunit in the dissociating gradients (panels A and B). This experiment provided three fractions for identification of the components crosslinked to the 4-thio-U in the mRNA: from the top of the subunit gradient, the fraction which would contain

FRACTION

NUMBER

Figure 3. The Crosslinking of 4-thioU-mRNA to ribosomal components. A,B. Association of crosslinked mRNA with ribosomal subunits. Ribosomal complexes formed with 70S ribosomes, [32p] labelled mRNA, tRNAala or thr, and RF-2 where indicated, irradiated at > 300nm, were sedimented through dissociating sucrose gradients (5-20% w/v). Sedimentation is from right to left.C,D. Association of crosslinked mRNA with ribosomal proteins or rRNA. The 30S peaks in A,B were isolated and sedimented in SDS/EDTA sucrose gradients(10-30% w/v). A,C.mRNA, tRNAala, no RF-2 (open circles), mRNA, tRNAala, RF-2 (closed circles). B,D. mRNA, tRNAthr, no RF-2(open circles) and mRNA? no tRNA (closed circles).

Nucleic Acids Research, Vol. 18, No. 22 6541 putative RF-2. mRNA (and tRNA.mRNA) complexes as well as the uncrosslinked mRNA, and from the SDS gradients an rRNA fraction, and a ribosomal protein fraction. Analysis of the crosslinked products (a) RF-2 The fraction containing putative RF-2 /mRNA crosslinked complexes also contained the mRNA which had not crosslinked to the ribosome and, therefore an SDS acrylamide gel system was used to resolve the [32p] labelled mRNA and the putative [35S] RF-2.[32P]mRNA complexes. The mRNA moves with the dye front and the [35S]RF-2 about a third of the way down the gel (Fig 4 A). If the mRNA and RF-2 were mixed before loading the gel there was no interaction between them. The samples analysed were from crosslinking experiments where the mRNA had been bound to the ribosome without tRNA and with tRNAala (both shown in panel B), and with RF-2 in the complex (C). In the sample bound without tRNA there was a large mRNA peak towards the bottom of the gel but little radioactivity in the positions of the putative crosslinked products (broken line). In contrast with tRNA there is a peak of radioactivity which we believe to be a tRNA.mRNA crosslinked product; indeed fragments of RNA containing the same number of nucleotides migrate to the same position, and tRNA alone migrates slightly faster than this peak in the gel system. Since the 4-thio-U is the nucleotide immediately proximal to the codon interacting with the tRNA such a crosslink might be expected. In the samples containing RF-2 this peak was depressed, and an additional [32p] labelled mRNA peak was seen in the position coincident with a [35S]RF-2 peak (C). The RF-2 molecule has a sensitive cleavage site and there was some degradation into two fragments during the crosslinking protocol (see the [35S] profile

3PRNA

A

21

in C). Difference plots between the experiments where RF-2 was present or absent ( an example is shown in D) showed nmore clearly the RF-2.mRNA complex. The [32p] mRNA was associated with the [35S]RF-2 reproducibly, and under conditions where the stability of the RF-2 ribosomal complex decreased this peak also decreased concomitantly in size. The 4-thio-U residue from which the crosslinking occurs is a 'zero length' moiety, and therefore the release factor and the codon must have been in close contact for the crosslink to occur.

(b) rRNA The mRNA/rRNA crosslinks were first analysed with a strategy involving pairs of deoxy ribo-oligonucleotides complementary to the rRNA, and subsequent RNaseH digestion (14). Where the crosslink is between the two deoxyribo-oligonucleotides it will be associated with the small fragment of the rRNA excised, while when it is outside this region it will remain with the larger rRNA fragments (see Fig 5C,D ). Several pairs of deoxyribooligonucleotides were used to identify the appproximate position of the crosslinks (1st step), and then they were narrowed to a few nucleotides in the rRNA by further RNase H digestion using new pairs of deoxyribo-oligonucleotides within the region in s

A

'B

S)

3 4

2

S

_MP _ .

.!%,jjjjjjjjjjm

q

No

B

41

35S RF2

4 0

0~~~~~~~~~

X D

I'

)-

/ t~~~~~/C'

4

v

_

(lanes 1.2)

R

t)

_

3|v .

(laness56)

0 ~~~~~~~~~ V

(lanes 3.4 )

LW.

0

20 10 FRACTION

0 NUMBER

10

20

Figure 4. SDS gel analysis to identify crosslinked mRNA/RF-2. Fractions isolated from the top of a dissociating sucrose gradient (see Fig 3A,B) were resolved by SDS gel electrophoresis. [32p] and [35S]radioactivity in gel slices were analysed. A. [31P] labelled mRNA and [35S] labelled RF-2 were mixed and resolved within the same gel lane as a control. B. The ribosomal complex containing mRNA, tRNAala but no RF-2 (o-o-o), and no tRNAala (----). C. The ribosomal complex containing mRNA,tRNAala and RF-2. D. A difference 1lot between gel profiles of samples containing and lacking RF-2 (C and B). [3 P]labelled mRNA-open circles; [35S]labelled RF-2-closed circles.

v

3.'p fragment

v

released

V

(lnes7.)

RNase FH cleavage site

Fige 5. Analysis of mRNA/rRNA crosslinks by RNase H digestion. Deoxyribooligonucleotide pairs were hybridised to the mRNA/rRNA crosslinked samples (see Fig 3C,D) and the complexes digested with RNase H. The products were resolved on polyacrylamide gels. Crosslinked samples where mRNA was bound to the ribosome with (A) tRNAala (crosslink A),and (B) tRNAthr (crosslink T). Lanes 1,5-undigested mRNA/rRNA; lanes 2,6 mRNA/rRNA sample hybridised with oligonucleotide pair near the 5'terminus of the rRNA; lanes 3,7 mRNA/rRNA sample hybridised with oligonucleotides spanning nucleotides - 1200 and -1400 respectively; lanes 4,8 mRNA/rRNA sample hybridised with oligonucleotides spanning - 1400 and - 1500 respectively. C,D provide schematic explanations for the patterns seen in A,B. The positions of the crosslinks (and hence the [32p] labelled mRNAs) are shown as hatched bars, and the oligonucleotide positions either as stars or with the nucleotide numbers. The arrows indicate the [32p] labelled fragments released in each case. In lane 7 there is only partial digestion with RNase H.

6542 Nucleic Acids Research, Vol. 18, No. 22 question (2nd step). Finally reverse transcriptase was used with primer extension from a position near the putative crosslink to identify the nucleotide in the rRNA crosslinked to the mRNA

(Fig 6).

In the samples derived from the mRNA bound to the ribosome with tRNAala a clear pattern emerged. The analysis with the first set of deoxyribo-oligonucleotide pairs and RNAse H digestion indicated that the crosslink was between nucleotides 1400-1500 towards the 3' terminus of the rRNA, a conclusion that could be drawn from two different sets of deoxyribooligonucleotides (Fig 5A lane 3 and 4). The size of the fragment in lane 3 (derived from a log plot of size vs migration) indicates the crosslink site has fallen 3' to the excised fragment of rRNA, that is within the 150 nucleotide 3' terminal fragment, whereas in lane 4 it is within the excised fragment of 100 nucleotides, that is the first part of the 3'terminal fragment (see Fig SC). In lanes 1 and 2 the undigested mRNA/rRNA sample and a digestion with another deoxyribo-oligonucleotide pair outside the crosslinked region are shown respectively. Here the crosslinked mRNA runs with the large rRNA (lane 1) or large rRNA -

-

-

'A

9

a

fragment (lane 2). The same result was obtained whether the RF-2 was present or not (see Fig SC for interpretation). In contrast a quite different result was obtained with the mRNA bound to the ribosome with the tRNAthr (Fig SB); now the crosslink was placed between nucleotides 1200-1400 and there was only partial excision of the fragment with RNaseH, illustrated by the two bands, suggesting that the crosslink site was near one of the deoxyribo-oligonucleotide binding positions (Fig SB lane 7). The same deoxyribo-oligonucleotide pairs that produced the 100 nucleotide rRNA fragment with the tRNAala bound mRNA gave a large fragment with the tRNAthr (lane 8) indicating that the crosslink site was 5' to this region. Lane 5,6 show the undigested RNA and the digestion with another deoxyribooligonucleotide pair, showing the mRNA associated with large rRNA fragments (see Fig SD for interpretation). The positions of the crosslinks were further defined with another set of deoxyribo-oligonucleotide pairs within the region of interest. In this case all experiments with the tRNAala gave similar results namely that the primary crosslink was between 1390-1415. Together with the information from Fig SA this

3*

_

0 '1*

1kr

AM,

As

46... I'A

---i

Figure 6. Identification of the mRNA /rRNA crosslinks by extension with reverse transcriptase. A deoxyribo-oligonucleotide hybridising with nucleotides 1492-1502 of the 16SrRNA was used with reverse transcriptase to extend to the crosslink sites where the mRNA was attached. In each case a sample from the same experiment but not containing the crosslink was a control. A. lane 1-control rRNA; lane 2 crosslinked mRNA/rRNA from ribosomal complex containing tRNAala and RF-2; lane 3-control rRNA, lane 4-crosslinked mRNA/rRNA from ribosomal complex containing tRNAala with no RF-2. lane 9-control rRNA; lane 10-crosslinked mRNA/rRNA from ribosomal complex containing tRNAt'u with no RF-2. Lanes 5-8 Extensions carried out in the presence of ddNTP's-lane 5-ddTTP; lane 6-ddGTP, lane 7-ddCTP, lane 8-ddATP. The sequence shown was derived and confirmned from multiple analyses. B. Secondary structure map of the 3' terminus of the 16SrRNA (11) showing the first set of oligonucleotides (i) used to obtain the approximate positions of the crosslink sites, the second set of oligonucleotides (ii) which narrowed down the positions of the sites, and the two sites (A- the tRNAala site, and T- the tRNAthr site) identified by extension with reverse transcriptase.

Nucleic Acids Research, Vol. 18, No. 22 6543 indicated that the site was likely to be between 1400-1415 both for the samples with or without RF-2. Similar experiments with tRNAthr gave the primary crosslink between 1390-1415. Together with data obtained in Fig SB this placed the crosslink site between 1390-1400. Extension was carried out with reverse transcriptase from an deoxyribo-oligonucleotide complementary to nucleotides 1492-1502 of the rRNA. The control for each sample was the uncrosslinked rRNA derived from the purification of the crosslinked samples. The reverse transcriptase has difficulty extending past certain positions in the rRNA, both because of the secondary structure of the RNA, modifications to the bases or nicks in the rRNA, and although the efficiency of traversing these sites is reproducible within a particular control sample, it can vary among different control samples.The presence of a crosslink in the vicinity of these natural stops may influence this efficiency, and therefore, while an enhanced stop should represent the crosslink site, a possible alternative explanation is an indirect effect from a nearby crosslink site. The extensions with the controls and their crosslinked samples are shown in lanes 1, 2 (tRNAala, RF-2), lanes 3, 4 (tRNAada, no RF-2), and lanes 9, 10 (tRNAthr). Sequencing reactions extending from the same deoxyribo-oligonucleotide are shown in lanes 5-8. In both tRNAala samples there was a more dominant stop corresponding to adenine 1408. This was reproducible in several crosslinked samples and triplicate analyses. The identified nucleotide falls within the region 1400-1415 suggested from the RNase H analyses. In the case of the tRNA'hr sample the crosslinked samples had a stop at adenine 1396 compared with its control. This is consistent with the RNase H analyses which suggested the crosslink was between nucleotides 1390-1400. In each of the lanes of the control samples there is a series of bands towards the top of the gel, characteristically lacking in the crosslinked samples. In Fig 6B the positions of the diagnostic deoxyribooligonucleotides -broad range (i) and narrow range(ii), and the crosslinks identified from the RNase H analyses and the reverse transcriptase extensions, are shown on the model for the secondary structure of the 3'terminal half of the 16SrRNA. Of significance is the position of the crosslink at 1396 which falls within the oligonucleotide binding region for one of the diagnostic deoxyribo-oligonucleotides. This may explain why there was incomplete RNase H cleavage of the rRNA in this sample (as shown in Fig SB lane7). We did not detect any new crosslink sites in the presence of RF-2, that is the crosslinking pattern from the termination codon to the rRNA did not change during codon recognition by the factor. However, if the codon was deliberately shifted by three nucleotides with respect to the ribosome a different pattern was observed. This result is consistent with at least one step of the decoding of the termination codon by the release factor occurring in the decoding site. We cannot eliminate the alternative possibility however that the factor is protecting the codon and not allowing crosslinking to rRNA when it is stably bound, and that the observed crosslinks in the presence of the factor result when the complex transiently dissociates. In the 3 dimensional model of the 30S subunit (11) nucleotide 1408 is at the end of helix 44 which extends up into the cleft, while 1396 is within the cleft but more towards the interface side of the subunit. Both of these positions are close to the decoding site and distant from helix 34 on the other side of the subunit where the major RF-2

binding domain is found (2). It should be noted they are within a long single stranded region of uncertain conformation, and that one model of the tertiary structure of the rRNA places helix 44 on the interface side of the subunit, and the other on the solvent side (11,17).

(b) ribosomal proteins The identity of ribosomal proteins containing covalently linked radiolabelled mRNA was determined by an immunological assay with antibodies specific for each protein. The protein in question is selected from the protein mixture by an agarose bound antibody and the radioactivity associated with it determined. Whether the ribosomal complex formed with tRNAala had originally contained RF-2 or not the patterns were indistinguishable; there were three proteins containing radiolabelled mRNA 2-3 fold above background, S1, S18 and to a lesser extent S21. The crosslink with S1 is not helpful to the analysis because the protein is large and covers a significant part of the subunit. However a crosslink with S18 and S21 when RF-2 is bound in the complex, would suggest that the factor is recognising the termination codon in the decoding site since these ribosomal proteins are placed in the vicinity of the cleft near the site. It is paradoxical however, that the crosslink to the rRNA was so specific and yet to the protein fraction was rather heterogeneous, although crosslinking from other mRNAs have given similar results (unpublished). When the crosslinking position was deliberately shifted by use of the tRNAthr to fix the mRNA on the ribosome then a somewhat different pattern of crosslinking was seen. While crosslinks to S1, S18, S21 were detected, other proteins, in particular S9, but also to a lesser extent S4, S12 and S14 also contained radioactivity associated with the mRNA above background levels. This implies there may be several proteins in relatively close proximity with the flexible mRNA as it approaches the decoding site which it could contact. The extent of crosslinking to any one protein was modest (up to 2-3 fold over background), in contrast that found for a mRNA when the crosslinking moiety was 5' to the decoding site position (leaving side); here S7 was the specific product with radioactivity 5-10 fold above background (12).

Implications for codon recognition There have been proposals that the termination codon might recognise a complementary sequence in the rRNA, and that the double stranded structure might be the template for the release factor (10,18). The position of the crosslink with the 16SrRNA is at a sequence 3' AmCU 5' which is complementary in two positions to UAA, and also in three positions with UGA, the two termination codons recognised by RF-2. The thioU in the modified codon should still be able to base pair with an A, although the geometry of the pairing would be somewhat altered

(19).

How do the conclusions from the studies described fit the various possible models of codon recognition? The features which differ are the proximity of factor and codon, the site on the 30S subunit for the recognition event, and whether the critical signal for termination is a double stranded structure which provides a binding domain for the release factor, or mediates a conformational change at a distant point allowing the factor to bind. Our studies are consistent with primary and direct recognition at the decoding site by the release factor, and they also provide an indication that base pairing of the codon and

6544 Nucleic Acids Research, Vol. 18, No. 22 16SrRNA could occur at the site, but we find no evidence for a significant conformational change when the termination complex forms. A second phase to recognition which our approach might not detect is still possible. This could be either a 'pre-reading' of the codon, perhaps involving mRNA/rRNA interaction before the main recognition event at the decoding site, or a second step of codon recognition which follows the primary event, perhaps involving a major conformational change and site change for the mRNA.This could be responsible for the peptidyl transferase converting from a 'peptide bond forming mode' to a 'polypeptide releasing mode' with water as the nucleophilic acceptor. Sitedirected crosslinking from free termination codons to RF and to ribosomal components, when the codons are not frozen by the tRNA complex, may help to resolve this question.

ACKNOWLEDGEMENTS The support of the Alexander von Humboldt Foundation, and the Medical Research Council of New Zealand to WPT is gratefully appreciated.

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