| Nucleic Acids Research | Pages |
Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit
Introduction
Materials And Methods
Materials
Methods
Results And Discussion
Non-covalent binding of 2[prime]-O-methyloligoRNAs to the 50S subunit
Site-specific photoincorporation of PHONT 1
Partial localization of regions in 23S rRNA labeled by PHONT 1 bound to its target site via azide-dependent photoincorporation, using RNase H cleavage
Identification of the major labeled site within the 2015-2305 fragment
Identification of the major labeled sites within the 2305-2501 fragment
Does 2258-48 probe binding to 50S subunits induce conformational change?
The `P-loop' subdomain and the peptidyl transferase center
Acknowledgements
References
Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit
Received August 11, 1999; Revised and Accepted September 30, 1999
ABSTRACT We report the synthesis of a radioactive, photolabile 2[prime]-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT 1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called `P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT 1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 Å from 23S RNA nucleotides G2252 and G2253. PHONT 1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.
INTRODUCTION
Elucidation of the mechanism of ribosome-catalyzed protein synthesis is a challenging task for contemporary biochemistry. Continuing efforts are focused on determining the structure of ribosomes (1-5) and on identifying key components involved in substrate recognition and catalysis of the peptidyl transferase reaction (6-14). Advances in cryoelectron microscopy have resulted in the publication of 15 Å resolution structures of 70S ribosomes, and structures at 10 Å resolution now appear feasible (1,2). Recent X-ray diffraction results have been even more dramatic, resulting in structures of the 30S and 50S subunits at 5.5 and 5.0 Å, respectively (3,4). However, even at these resolutions, the precise placement of ribosomal components within the ribosome structure can be a daunting task. Chemical crosslinking can provide information that is helpful in this regard.
We have been using radioactive photolabile oligonucleotide probes (PHONTs) targeted toward functionally important rRNA sites to identify proteins and individual nucleotides site-specifically photolabeled from these sites as a way of generating crosslinking information useful for the development of detailed structural models (15-21). The PHONT approach offers several important advantages for such an exercise. First, it allows the targeting of sequences of particular functional or structural significance throughout the ribosome structure. Second, it is readily applicable to the examination of the nature of the substantial conformational changes that the ribosome undergoes during the overall cycle of protein biosynthesis. Third, the crosslinks formed provide defined upper limit distances for the separation of the linked components within the ribosome, given by the length of the tether arm. As the length of this arm can be varied, the approach can be used to identify components both immediately neighboring the target site or somewhat further away. Fourth, sample preparation employing readily synthesized PHONTs and intact ribosomes is straightforward; an important attribute given the large number of constraints required for model building. More general considerations of the PHONT approach, which can be applied to study any complex RNA-containing molecule (e.g. spliceosomes, RNase P and ribozymes), are presented by Cooperman et al. (22,23).
Here we target Escherichia coli 23S rRNA nt 2248-2258. These nucleotides fall within the so-called `P-loop' (nt 2246-2258) that is a vital part of the peptidyl transferase center. It contains three invariant guanosine residues, G2251-G2253, of which two, G2252 and G2253, are protected from kethoxal modification by P-site bound tRNA (24). In addition, mutational analysis has shown that G2252 forms a Watson-Crick pair with C74 of the P-site bound tRNA (25,26); mutations at G2251 abolish both binding of wild-type tRNA fragments and peptidyl transferase activity (27); and there is evidence for direct or indirect interaction of the 2250-loop with U2585, which falls within the central loop of domain V that defines another part of the peptidyl transferase center (28,29). Specifically, we use PHONT 1 (Fig. 1), a 2[prime]-O-methyloligoRNA complementary to nt 2248-2258 and containing a p-azidophenacyl group attached to a phosphorothioate bridging nucleotide complementary to nt 2252-2253, to identify ribosomal components within 17-19 Å of these nucleotides.
Figure 1. PHONT 1. The distances between the photogenerated nitrene and the 2-amino positions of the complemented nt G2252 and G2253 in 23S rRNA are 17-19 Å.
MATERIALS AND METHODS
Except as specified below, all materials were obtained and all methods were carried out as described previously (15,18,20,22). 50S subunits were activated at 37°C for 20 min in the presence of 10 mM Mg2+ immediately prior to the binding and photocrosslinking experiments.
Materials
All oligonucleotides were synthesized using phosphoramidite chemistry on a Milligen Biosearch Cyclone automated DNA synthesizer.
Synthesis and purification of PHONT 1, 2[prime]-O-methyloligoRNA 2258-53/2(SAz)-48 (Fig. 1)2[prime]-O-methyloligoRNA 2258-53/2(S)-48 (0.1 µmol), gaccgC(S)cccag, which contains a phosphorothioate group bridging the sixth and the seventh nucleotides, was reacted with 2 µmol of 4-azidophenacylbromide (Sigma) in 50 µl of 100 mM sodium bicarbonate containing 50% dimethylsulfoxide (DMSO). Reaction was allowed to proceed at room temperature for 1.5 h, after which the mixture was diluted with water to 160 µl and extracted twice with 300 µl of isobutanol. The product, PHONT 1, was purified by reverse phase HPLC on an octadecylsilica column (SynChropak RP-P, MICRA Scientific, Inc.) using a linear gradient from 5 to 40% acetonitrile in 100 mM triethylammonium acetate, pH 7.4. Starting material and PHONT 1 eluted as two pairs of diastereo-mers, with the product pair eluting later. Both isomers of the product were collected and used in equal amounts for the photoaffinity labeling experiments. The corresponding phenacyl derivative, 2[prime]-O-methyloligoRNA 2258-52/3(S[Phi])-48, was prepared in a similar manner by using bromoacetophenone (Sigma) as alkylating reagent.
Figure 2. Non-covalent binding of 32P-2[prime]-O-methyloligoRNAs to 50S subunits. PHONT 1 (circle); 2[prime]-O-methyloligoRNA 2258-48 (diamond); 2[prime]-O-methyloligoRNA 2255-49 (square). The nitrocellulose filter binding assay was performed as described in Materials and Methods.
Methods
Non-covalent binding of oligonucleotides to 50S ribosomal subunit. 50S subunits (15 pmol) were combined with varying amounts of 32P-2[prime]-O-methyloligoRNA in the presence of 40 mM Tris-HCl (pH 7.6), 60 mM KCl and 0.45 mM MgCl2 in a total volume of 25 µl. The mixture was incubated at 37°C for 5 min and on ice for 15 min. The MgCl2 concentration was then raised to 10 mM, and the mixture was incubated at 37°C for 5 min and on ice for 2 h. The samples were diluted with 0.5 ml of buffer and applied onto Millipore 0.45 µm nitrocellulose membrane filters type HAWP that had been pre-washed with 5 ml of the binding buffer containing 10 mM MgCl2. The filters were washed three times with 1 ml of the buffer, air-dried and counted for radioactivity in EcoLite scintillant. Binding measurements were performed in triplicate.
Photoaffinity labeling of 50S ribosomal subunit and isolation of labeled 23S rRNA. PHONT 1 (300 pmol) was radiolabeled at the 5[prime]-end with 250 µCi of [[gamma]-32P]ATP using T4 polynucleotide kinase (Promega) (30). The radiolabeled probe was purified by solid-phase extraction on a Sep-Pak C18 cartridge (30) and recovered in ~50% yield. The product was divided into four equal amounts, and each was incubated with 150 pmol of 50S subunit in 75 µl of T50K50M0.3 buffer (50 mM Tris-HCl, pH 7.6, 50 mM KCl, 0.3 mM MgCl2) at 37°C for 5 min and left on ice for 15 min. The Mg2+ concentration was increased to 10 mM and the samples were incubated at 37°C for 5 min and kept on ice for 30 min. The solutions were irradiated in 250 µl Pyrex glass inserts for autosampler vials (Kimble/Kontes) at 4°C for 4 min using 3000 Å mercury lamps (Rayonet). The ribonucleoprotein complex was precipitated with 2 vol of 10% 2-mercaptoethanol in ethanol at 4°C, and the precipitate was washed with cold 70% ethanol. The dried pellet was dissolved in 24 µl of T50K50M10 buffer (50 mM Tris-HCl, pH 7.6, 50 mM KCl, 10 mM MgCl2), and 23S rRNA was precipitated by addition of 67 µl of 100 mM MgCl2 in 90% acetic acid (0°C, 45 min). The procedure was repeated, and the dried pellet was dissolved in 100 mM sodium acetate, pH 5.2, extracted with phenol, phenol-chloroform and chloroform, and precipitated overnight at -20°C with 2 vol of ethanol. Samples prepared for reverse transcriptase assays were photolyzed with non-radioactive PHONT 1 at higher probe/subunit ratios (10:1 or 20:1).
RNase H cleavage of [32P]PHONT 1-labeled 23S rRNA. Five picomoles of the RNA was hybridized with 10 pmol of oligodeoxyribonucleotides or DNA/2[prime]-O-methylRNA hybrid oligonucleotides by heating at 55°C for 5 min and incubating at 32°C for 15 min in 8 µl of T25N125D1.25 buffer (25 mM Tris-HCl, pH 7.6, 125 mM NaCl, 1.25 mM DTT). Mg2+ was adjusted to 10 mM and the RNA was digested with 2 U of RNase H (Promega) for 30 min at 32°C. To these samples 8 µl of 95% formamide containing 50 pmol/µl of 2[prime]-O-methyloligoRNA 2258-48, 0.1% of bromophenol blue and xylene cyanol was added, the samples were heated at 65°C for 10 min and separated on an acrylamide-7 M-urea 90 mM Tris-borate, pH 8.3, 2 mM EDTA gel (the acrylamide varied between 5 and 10% depending on the desired size range) preheated to 45°C by applying a 20-25 mA current. The added excess complementary unlabeled parent oligonucleotide reduced background due to non-covalent PHONT 1 in the urea-PAGE analysis.
Sequence scanning. Sequence scanning was carried out using AMPLIFY (authored by Bill Engels, www.wisc.edu/genestest/CATG/amplify ).
Reverse transcriptase primer extension. The procedure employed followed Muralikrishna and Wickstrom (31). Typically, 5[prime]-32P-labeled primer oligoDNA (0.5-1 pmol, 1.5 × 106 c.p.m., 3 µl), prepared using T4 polynucleotide kinase as described above, was annealed to rRNA (0.6 pmol, 1 µl) by incubation in 50 mM Tris-HCl (pH 8.3), 60 mM NaCl and 10 mM DTT for 5 min at 70°C. Samples were frozen in isopropanol/dry ice bath (1 min) and then kept on ice for at least 30 min, after which time they were microfuged briefly to bring down any liquid from the sides of the tube. To each annealing mix was added extension mix (6 µl), containing 10 U of avian myeloblastosis virus (AMV)-reverse transcriptase (Promega) and affording the following final concentrations in 10 µl total volume: each dNTP (0.32 mM), 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 10 mM DTT and 0.5 mM spermidine. For sequencing samples, one ddNTP was added to a final concentration of 0.16 mM. Samples were incubated at 47°C for 30 min then quenched by addition of 5 µl of 100% formamide/0.1% xylene cyanol FF/0.1% bromophenol blue loading buffer. After 10 min at 70°C, samples were loaded on an 6% polyacrylamide urea sequencing gel and electrophoresed at 1400-1500 V for 1-1.5 h until bromophenol blue reached the bottom of the gel. The temperature of the gel during the run was maintained at 45-55°C.
Chemical footprinting. The procedure followed Moazed et al. (32) with slight modifications. Samples (75 µl) containing 50S subunits (150 pmol) either in the absence or presence (3-10-fold molar excess) of complementary oligonucleotides were incubated with either dimethyl sulfate (DMS) (1 µl) or kethoxal (8 µl of 37 mg/ml kethoxal in 20% ethanol) at 4°C for 2.5-3 h with gentle shaking. Reactions were stopped by addition of either 75 µl of 1 M Tris-acetate (pH 7.5), 1 M 2-mercaptoethanol, 1.5 M NaAc and 0.1 mM EDTA (DMS reaction) or 20 µl of 0.5 M potassium borate (pH 7.0) (kethoxal reaction). Following incubation on ice for 10 min, subunits were ethanol precipitated, washed with 70% ethanol and rRNA was extracted by phenol-chloroform extraction for analysis by reverse transcriptase primer extension.
RESULTS AND DISCUSSION
Non-covalent binding of 2[prime]-O-methyloligoRNAs to the 50S subunit
The 11 residue 2[prime]-O-methyloligoRNA 2258-48 binds non-covalently to 50S subunits in a biphasic manner, with a high affinity site stoichiometry of ~0.4/50S subunits, and a second class of sites of lower affinity (Fig. 2). The shorter 2[prime]-O-methyloligoRNA 2255-48 shows a lower high affinity site stoichiometry (~0.1/50S) and no evidence of lower affinity site binding. The higher binding stoichiometry of the longer probe led us to design the photolabile 2[prime]-O-methyloligoRNA 2258-52/3(SAz)-48, PHONT 1, in which the phosphorothioate between nucleotides complementary to G2252 and G2253 is derivatized with an azidophenacyl group. PHONT 1 binds to 50S subunits in a biphasic manner similar to its parent 2[prime]-O-methyloligoRNA, but with a reduced high affinity site stoichiometry (~0.2/50S).
Site-specific photoincorporation of PHONT 1
Photolysis of non-covalent complexes of 50S subunits with PHONT 1 were generally performed at excess 50S versus PHONT 1 (for an exception see below), in order to maximize photoincorporation from the high affinity site. Evidence for site-specific crosslinking was sought by comparison of results obtained with PHONT 1 alone, and with PHONT 1 in the presence of either excess competing 2[prime]-O-methyloligoRNA 2258-48, GACCGCCCCAG, which should show a large reduction in photoincorporation, or of the mismatched oligonucleotide, GACAC-AACCAG (mismatched bases are underlined), which should show little effect on photoincorporation. Although both reverse phase high performance liquid chromatography and SDS-PAGE analysis showed photoincorporation into 50 S proteins, neither analysis provided unambiguous evidence for specific labeling of any protein (data not shown). On the other hand, 23S rRNA, but not 5S rRNA, was site-specifically labeled, as described below.
Partial localization of regions in 23S rRNA labeled by PHONT 1 bound to its target site via azide-dependent photoincorporation, using RNase H cleavage
Partial localization of the photo-crosslinking sites was achieved by RNase H cleavage of the labeled 23S rRNA hybridized with a series of deoxyoligonucleotides (Table 1) that, used together, generated fragments ~150-400 nt long over the whole length of 23S rRNA. Fragments excised by RNase H were analyzed by urea-PAGE and autoradiography (Fig. 3). The highest labeling of 23S rRNA was found in fragments 2015-2305, which includes the targeted sequence 2248-2258, and 2305-2501, the 3[prime]-neighboring sequence. This labeling was site-specific, as shown by the results obtained in the presence of complementary or mismatched 2[prime]-O-methyloligoRNA. No other radiolabeled fragments were observed in these experiments. An example of the characteristic negative results seen with the regions of the 23S rRNA other than between 2015 and 2501 is shown in Figure 3, lanes 1675-1882.
Figure 3. RNase H cleavage of [32P]PHONT 1-labeled 23S rRNA using oligo DNAs. Photolabeling experiments were carried out with an ~4-fold excess of 50S subunits over [32P]PHONT 1 either in the absence of added 2[prime]-O-methyloligoRNA [lanes (-) and (+)], or in the presence of a 10-fold excess over 50S subunits of 2[prime]-O-methyloligoRNA 2248-58 (GACCGCCCCAG) (lanes CH) or of mismatched 2[prime]-O-methyloligoRNA 2248-58 (GACACAACCAG, mismatched bases are underlined, lanes MM). Asterisks over the bar at top indicate the major labeled fragments: 2015-2305 and 2305-2501. The (-) lanes represent `dark reaction' control experiments in which PHONT 1 was pre-irradiated for 4 min at 4°C prior to dark incubation with 50S subunits, carried out as described in Materials and Methods. Lanes C, no nucleotides were hybridized to 23S rRNA during RNase H digestion. In other lanes, RNase H digestion followed hybridization with the following added cDNAs: 2501, 2505-2497; 2305-2501, 2310-2301 and 2505-2497; 2015-2305, 2020-2011 and 2310-2301; 1675-1852, 1680-1671 and 1857-1848. Numbers in the left margin indicate sizes (in nt) of DNA markers.
Table 1. Oligonucleotides used for RNase H cleavage of 23S rRNA
| Oligonucleotides for partial localization | Sequencea | Oligonucleotides for more precise localization | Sequencea |
| 222-213 | TTCTTTTCCT | 2333-2324 | UGCCATTGCA |
| 455-447 | GTTCACTAT | 2355-2346 | CGCTCGCAGU |
| 677-669 | TTTCGGGTC | 2360-2351 | CGTCACGCUC |
| 811-803 | AACCAGCTA | 2374-2365 | GCACCUGCTC |
| 1051-1042 | CTGTCTGGGC | 2379-2370 | CUUTCGCACC |
| 1274-1265 | TATCGTTACT | 2380-2371 | GCTTUCGCAC |
| 1460-1451 | ACAACCGTCG | 2400-2391 | CCACCGGAUC |
| 1680-1671 | ATTTTGCCTA | 2413-2404 | CUUCCATUCA |
| 1857-1848 | CAATTAACCT | 2431-2422 | AUCCGTTGAG |
| 2020-2011 | TTCAATTTCA | 2450-2441 | TATCCCCGGA |
| 2230-2221 | CACTGTCCGC | 2460-2451 | ATCAGCCUGU |
| 2310-2301 | GTCCGACCAG | 2500-2491 | AGGTGCCAAA |
| 2505-2497 | CATCGAGGU | 2505-2497 | CATCGAGGU |
It is important to verify that photoincorporation into regions 2015-2305 and 2305-2501 arises from PHONT 1 bound to its target site, rather than from a `fortuitous' site, defined as a sequence within 23S rRNA having substantial (>7 out of 11 nt) sequence overlap with nt 2248-2258. Accordingly, 23S rRNA was scanned for possible `fortuitous' sequences. Five were found; two with three mismatches versus 2248-2258 (2521-2531 and 2636-2646) and three with four mismatches (772-782, 1158-1168 and 1512-1522). 2[prime]-O-methyloligoRNAs complementary to these five sequences were then compared with the 2258-48 2[prime]-O-methyloligoRNA for their effects on PHONT 1 photoincorporation into regions 2015-2305 and 2305-2501. The results (Fig. 4) clearly show that none of the five `fortuitous' oligonucleotides are as effective as the target site oligonucleotide in inhibiting PHONT 1 photoincorporation into regions 2015-2305 and 2305-2501.
Figure 4. [32P]PHONT 1 photoincorporation into fragments 2015-2305 and 2305-2501. Comparative effects of added target site and `fortuitous' site 2[prime]-O-methyloligoRNAs. Procedures were as described for Figure 3. Photolabeling experiments were carried out either in the absence of added 2[prime]-O-methyloligoRNA (lanes 1), or in the presence of the indicated concentration of 2[prime]-O-methyloligoRNA 2248-58 (lanes 2-5), or of 2[prime]-O-methyloligoRNAs, added at 20 µM, that are complementary to the `fortuitous' sites 2531-2521 (UUCAGCCCCAG, lanes 6), 2646-2636 (GCCCCCCUCAG, lanes 7), 1522-1512 (UCACGCCUCAG, lanes 8), 1168-1158 (CGCUGCCGCAG, lanes 9) and 782-772 (UUCACCCCCAG, lanes 10). (A) Fragment 2015-2305; (B) fragment 2305-2501. Arrows in the right margin indicate migrations of DNA size markers.
To verify that photo-crosslinking to regions 2015-2305 and 2305-2501 is azide specific, we compared photoincorporation results obtained with 32P-labeled PHONT 1 with those obtained with 32P-labeled samples of (a) 2[prime]-O-methyloligoRNA 2258-48, (b) 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 and (c) 2[prime]-O-methyloligoRNA 2258-52/3(S[Phi])-48. The three standard photoincorporation experiments shown above [(i) photolysis experiment, (ii) plus added excess complementary 2[prime]-O-methyloligoRNA-2258-48 and (iii) plus added mismatched oligonucleotide; see Fig. 3] were carried out for each of these four oligonucleotides. Experiments were also carried out with samples that had been pre-photolyzed prior to incubation in the presence of 50S subunits to test for possible `dark reaction' occurring between the photo-activated oligonucleotides and the targeted region on 23S rRNA. From Figure 5B it is clear that of the four oligonucleotides only PHONT 1 shows significant photoincorporation into region 2305-2501. The lack of photoincorporation of 2[prime]-O-methyloligoRNA 2258-52/3(S[Phi])-48 provides unambiguous evidence that photoincorporation within nt 2305-2501 is azide-dependent.
Figure 5. RNase H cleavage of the 23S rRNA photolyzed in the presence of four [32P]2[prime]-O-methyloligoRNAs complementary to nt 2258-2248. Procedures and lane definitions are as in Figure 3. Lanes 2258-48, 2[prime]-O-methyloligoRNA 2248-58; lanes 2258-(S)-48, 2[prime]-O-methyloligoRNA 2258-53/2(S)-48; middle lanes, DNA and RNA ladders; lanes 2258-(S[Phi])-48, 2[prime]-O-methyloligoRNA 2258-53/2(S[Phi])-48; and lanes PHONT 1, 2[prime]-O-methyloligoRNA 2258-53/2(SAz)-48. (A) Fragment 2015-2305; (B) fragment 2305-2501. Arrows in the right margin indicate migrations of DNA size markers.
The situation is less clear-cut in region 2015-2305 (Fig. 5A). While PHONT 1 gives by far the largest extent of site-specific photoincorporation, all three other oligonucleotides also show apparent site-specific photoincorporation, reaching levels as high as 25-30% (as determined by autoradiogram scanning) of that obtained for PHONT 1. It is probable that some of such `photoincorporation' arises from one or more non-azide photo-dependent chemical reactions along the entire length of the oligonucleotide bound to its target site. In addition, some of the observed incorporation probably reflects incomplete removal of non-covalently bound material, since, as is clear from the lanes labeled (-), pre-photolyzed PHONT 1 [and pre-photolyzed 2[prime]-O-methyloligoRNA 2258-52/3(S[Phi])-48 as well] shows a lower incorporation level than 2[prime]-O-methyloligoRNA 2258-48, in accord with the relative binding stoichiometries seen in Figure 2.
Identification of the major labeled site within the 2015-2305 fragment
Evidence that labeling in the 2015-2305 region takes place dominantly between nt 2225 and 2305 is provided by the generation of a 32P-labeled fragment of ~85 nt in length when PHONT 1-labeled 23S rRNA is digested with RNase H in the presence of the cDNA probes 2230-2221 and 2310-2301. In addition, the same digestion carried out in the presence of the cDNA probes 2020-2011 and 2230-2221 fails to generate a 32P-labeled fragment of ~200 nt in length (data not shown). Identification of the major labeled site within the 2230-2221 to 2310-2301 fragment was accomplished using 23S rRNA isolated from PHONT 1-labeled 50S as a template for AMV-reverse transcriptase. In these experiments, the ability to detect a PHONT 1-dependent stop or pause (below, for simplicity, we use stop to indicate stop or pause) depends on the stoichiometry of incorporated PHONT, unlike the RNase H experiments where even quite low levels of incorporation are detectable due to the high specific radioactivity of the 32P-labeled PHONT. Accordingly, we used a 10-fold molar excess of (non-radioactive) PHONT 1 over 50S subunits. Only one light- and PHONT 1-dependent stop was seen within the 2210-2352 region, at C2260, corresponding to photoincorporation into U2259, immediately adjacent to the target site (Fig. 6). This result is an important demonstration that PHONT 1 binds to its target site during photolysis, but provides no structural information for the 50S subunit.
Figure 6. Primer extension analysis of PHONT 1-labeled 23S rRNA. The 2352-2210 region. All reaction mixtures contained 0.15 nmol of 50S subunit, and the indicated quantity (in nmole) of PHONT 1. Photolysis was as indicated. RNA was prepared for primer extension as described in Materials and Methods. The primer used was oligo-DNA 2493-2477. Lanes U, C, G and A are sequencing products generated from control 23S rRNA in the presence of ddATP, ddGTP, ddCTP and ddTTP, respectively.
Identification of the major labeled sites within the 2305-2501 fragment
Identification of photoincorporation sites within this fragment is obviously of greater interest for defining the structure of the peptidyl transferase center. For this purpose we modified the RNase H procedure to allow more precise localization of labeled nucleotides, and utilized primer extension by reverse transcriptase as well.
In the experiments presented in Figures 3-5 we used oligoDNAs to create RNase H sites, with the result that the RNA fragments excised were microheterogeneous, since RNase H cuts at several places within each oligoDNA-23S rRNA heteroduplex. Such heterogeneity was not a problem for localization of labeling within >100 nt or so, but more precise localization requires a more precise cutting tool. This was provided by hybrid oligonucleotides containing four con-secutive deoxynucleotides with the remainder of the residues being 2[prime]-O-methyloligo-ribonucleotides. Such molecules, hybridized to 23S rRNA, create unique RNase H cutting sites (33), resulting in excised RNA fragments of precise length. Application of this approach to 50S subunits photolabeled with 32P-PHONT 1 showed that photoincorporation occurs at four locations in this region, all falling between nt 2355 and 2450 (Fig. 7). The first, occurring within 2330-2376 (lane 4) can be more precisely localized to within nt 2355-2360 by the absence of a labeled fragment in lanes 1 (2330-2355) and 6 (2360-2376) and the presence of such a fragment in lanes 2 (2330-2360) and 5 (2355-2376). Two other labeled sites can be localized between 2376 and 2400 (lane 7) and between 2400 and 2428 (lane 10). Finally, there is a fourth site between 2428 and 2450, as shown by the presence of labeled bands in lanes 11 (2428-2450) and 12 (2428-2460), and the absence of a labeled band in lane 15 (2460-2505). It is important to emphasize (see below) that all four of these sites are labeled site-specifically, since added 2[prime]-O-methyloligoRNA-2258-48 completely suppresses PHONT 1 photoincorporation into the 2305-2501 fragment (Fig. 3).
Figure 7. RNase H cleavage within fragment 2305-2501 of [32P]PHONT 1-labeled 23S rRNA using hybrid oligonucleotides. Procedures were as described for Figure 3. PHONT 1-labeled rRNA (5 pmol) was hybridized with the indicated pairs of DNA/2[prime]-O-methyloligoRNA hybrid oligonucleotides (10 pmol each, see Table 1), digested with 2 U of RNase H and separated on a 10% acrylamide-urea gel. Note that labeled fragments migrate with apparent sizes (in nt) equal to the sum of excised fragment and PHONT 1 (11 nt). Numbers in the left and right margins indicate sizes (in nt) of DNA markers.
Primer extension (Fig. 8, lane 4) of PHONT 1-labeled 23S rRNA shows stops at seven positions (1, A2358; 2, C2359; 3, G2405; 4, A2406; 5, A2407; 6, A2412; and 7, U2431) that are not seen in 23S rRNA isolated from (i) control 50S subunits (lane 1), (ii) 50S subunits incubated with PHONT 1 in the absence of irradiation (lane 2), (iii) 50S subunits irradiated in the absence of PHONT 1 (lane 3) or (iv) 50S subunits irradiated in the presence of the non-photolabile 2[prime]-O-methyloligoRNA 2258-2252/3(S)-2248 (lane 5). In the data shown (Fig. 8), cDNA complementary to nt 2457-2442 was used as primer, but similar results were obtained using cDNA complementary to nucleotides 2493-2477 and 2443-2427. Stops 1 and 2, 3-6, and 7 are fully consistent with the site-specifically labeled sites 2355-2360, 2400-2428 and 2428-2450, respectively. No stop is seen within labeling site 2376-2400, but PHONT 1-dependent stops within this site would be difficult to detect because of the large number of stops seen in control 23S rRNA.
Figure 8. Primer extension analysis of PHONT 1-labeled 23S rRNA. The 2434-2290 region. All reaction mixtures contained 0.06 nmol of 50S subunit, and the indicated quantity (in nmole) of 2[prime]-O-methyloligoRNA. Photolysis was as indicated. The primer used was oligo-DNA 2457-2442. Lanes U, G, C and A are sequencing products generated from control 23S rRNA in the presence of ddATP, ddCTP, ddGTP and ddTTP, respectively.
Despite this gratifying consistency between the two sets of results, there is also an apparent inconsistency that we do not as yet fully understand. In contrast to the suppression of labeling of the 2305-2501 fragment by added 2[prime]-O-methyloligoRNA 2258-48 (Fig. 3), added non-photolabile 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 (or added 2[prime]-O-methyloligoRNA 2258-48, data not shown), in ratios of 1:1, 2.5:1 and 5:1 with respect to PHONT 1 (Fig. 8, lanes 6-8), does not markedly reduce the intensities of the stops at positions 1-7.
The most important difference between the two experiments is in the ratio of PHONT 1/50S subunits during photolysis, which was 0.25 and 10 in the experiments using RNase H digestion (Fig. 7) and primer extension (Fig. 8), respectively. Considering both this difference and the heterogeneity in binding noted in Figure 2, a plausible explanation for the apparent inconsistency can be advanced, based on three reasonable assumptions: (i) at a low PHONT 1/50S ratio, PHONT 1 binds to its target site only in high-affinity subunits, whereas at a high ratio it binds to this site in both high and low affinity 50S subunits; (ii) both types of 50S-PHONT 1 complexes form photocrosslinks to the same nucleotides within 23S rRNA but, at a PHONT 1/50 S ratio of 10, the overall photocrosslink yield is higher in the low affinity ribosomes; and (iii) the concentration of added complementary oligonucleotide is sufficient to block labeling in the high-affinity complexes but not in the low-affinity complexes. Further experiments will be required to test the validity of this explanation. We note that high concentrations of oligonucleotides might introduce artifacts into primer extension analysis (i.e. stops or pauses not directly arising from PHONT 1 photoincorporation) that could also contribute to the apparent inconsistency.
Summarizing, PHONT 1 site-specifically photoincorporates into four sites in 23S rRNA, nts 2355-2360, 2376-2400, 2401-2428 and 2428-2450, and there is evidence, albeit somewhat ambiguous, that such photoincorporation reflects labeling of nts G2357, A2358, U2404, G2405, A2406, A2411 and A2430 as well as an unidentified site (or sites) within nts 2376-2400.
Does 2258-48 probe binding to 50S subunits induce conformational change?
Since we seek to generate photocrosslinks that will be useful in constructing and evaluating models of the peptidyl transferase center within the 50S subunit, it is important to assess whether PHONT 1 binding to the 50S subunit causes structural distortions outside of the expected local distortion at its binding site. For this purpose we examined (Fig. 9) the effect of binding of 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 on chemical footprinting (DMS and kethoxal) patterns in regions of 23S rRNA [nt 750-1130, 1780-2215 and 2258-2650; the gap is caused by the binding of 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 to nt 2248-58] that either include the crosslinking sites described above or that have been implicated either directly or indirectly in peptidyl transferase activity (5-13). Most significantly, added 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 has no effect on the chemical modification of nt 2258-2650, which includes the region from 2350 to 2435 (Fig. 9A) and that contains all of the nucleotides into which PHONT 1 photoincorporates site-specifically. Thus, none of this photoincorporation can be attributed to distortions in the 23S rRNA structure.
Figure 9. Primer extension analysis of the effect of 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 on chemical modification of 23S rRNA within 50S subunits. (A) Lanes U, G, C and A are sequencing products generated from control 23S rRNA in the presence of ddATP, ddGTP, ddCTP and ddTTP, respectively. Sequencing lanes are omitted from other panels for clarity. Lanes (-) and (+) denote the absence or presence of 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 during incubation of 50S subunits; lanes UNMOD, DMS and KETH denote incubations of 50S subunits in the absence of chemical modification, in the presence of DMS or in the presence of kethoxal, respectively. The oligo-DNA primers used for extension were as follows. (A) 2457-2442. (B) 1115-1099. (C) 2125-2109. (D) 2250-2234. Arrows indicate major sites of 2[prime]-O-methyloligoRNA 2258-53/2(S)-48-induced change, including the target region for 2[prime]-O-methyloligoRNA 2258-53/2(S)-48 in (A). Double and triple arrows indicate stops at consecutive nucleotides. Arrows are aligned with stops in DMS lanes.
Similarly, no changes were seen for nt 750-970 (data not shown). However, a series of changes were seen in regions 990-1057 (Fig. 9B), 2005-2019 (Fig. 9C) and 2169-2212 (Fig. 9D), as well as at nt A1927 and A1928 (data not shown) and A2071, A2088 and A2090 (Fig. 9C). Interestingly, each of the changes depicted in Figure 9B-D are also induced by the binding of other PHONTs directed toward the peptidyl transferase center, targeting nt 2448-2458 and 2604-2612. As discussed in greater detail elsewhere (S.N.Vladimirov, Z.Druzina, R.Wang and B.S.Cooperman, submitted for publication), these changes are likely to arise from some combination of conformational change induced by complementary oligonucleotide binding to the 50S subunit and of physical proximity between the PHONT-targeted sites and nucleotides showing altered reactivity.
The `P-loop' subdomain and the peptidyl transferase center
The crosslinks we observed (Fig. 10) demonstrate a physical proximity (17-19 Å) between nt 2252 and 2253 within the P-loop of 23S rRNA (attached to helix 80), a key part of the binding site for the 3[prime]-terminus of P-site bound tRNA (see above), and four other sites within region V of 23S rRNA falling between (i) nt 2355 and 2360 (most likely nt G2357 and A2358) in the loop attached to helix 86, (ii) nt 2376 and 2400, in the region defined by helices 87 and 88, (iii) nt 2400 and 2428 (most likely U2404-A2406 and A2411) a region including helix 88 and its attached loop and (iv) nt 2428 and 2450 (most likely A2430) falling between helices 74 and 88.
Figure 10. Identification of crosslinks. The portion of Domain V of the 23S rRNA showing major labeling by PHONT 1. Arrows point to crosslinks identified by primer extension analysis.
These results show a remarkable overlap with those of Gregory and Dahlberg (34), who have demonstrated that mutations within the P-loop (at nt G2251, G2252 or G2253) result in major enhancements in the chemical reactivities of several nucleotides within 70S ribosomes, including G2357, G2361, U2408-U2410, G2428 and U2431, as well as with a direct crosslink between nt 2258 and 2425 previously reported by Stiege et al. (35). They thus strongly buttress the conclusion of Gregory and Dahlberg (34), based as well on phylogenetically-determined tertiary interactions (36), that the P-loop forms part of a structural subdomain extending from G2238 to A2433.
On the other hand, we did not see any crosslinks to either the 2530 loop, the A loop (U2552-C2556) or the 2585 region in 23S rRNA, although mutations within the P-loop induce altered chemical reactivities in each of these 23S rRNA sites (27,34). This may indicate that the latter changes arise from allosteric effects, rather than from the direct physical proximity we demonstrate between the P-loop and nucleotides falling between 2357 and 2430. Alternatively, it is possible that the orientation of the azido group within the heteroduplex formed between the RNA and PHONT 1 does not allow crosslinks to these other sites to be formed, since modeling the heteroduplex structure makes clear that only a limited region of the 50S subunit is accessible to the photogenerated nitrene. Such an explanation might also account for the lack of labeling of L27, which has been crosslinked to several nucleotides in the P loop subdomain (37). Here it is worth noting that the distance between the phosphate backbone of the photoprobe and the photogenerated nitrene nitrogen is only 8.9 Å. Further, attachment of the phenacyl moiety in the middle of the oligonucleotide, as opposed to either the 3[prime]- or 5[prime]-termini, limits its freedom of motion. It would be interesting to determine whether other PHONTs targeted to the 2250 loop will crosslink within the 50S subunit at sites other than those described above, following the logic of experiments recently carried out on the 530 loop of 16S rRNA (21).
There is abundant evidence that the ribosome is a flexible structure, undergoing large-scale conformational changes as it proceeds from one functional state to another (38,39). As X-ray structures achieve increasingly fine resolution, we expect that PHONT and other site-directed photo-crosslinking studies (40,41) will change focus, from generating photocrosslinks to aid in the construction of a static ribosome structure to using crosslinks as a way of defining conformational changes occurring in different functional states of the dynamic ribosome. Here, the intrinsic ability of the PHONT approach to sample available conformations in solution from functionally significant targeted sequences will be of particular utility.
ACKNOWLEDGEMENTS
We acknowledge with thanks the excellent technical assistance of Nora Zuño in several aspects of this work and Hyuk-Soo Seo for his help with preparation of the manuscript. This work was supported by NIH grant GM-53146.
REFERENCES
*To whom correspondence should be addressed. Tel: +1 215 898 6330; Fax: +1 215 898 2037; Email: cooprman{at}pobox.upenn.edu Present address: Yuri Bukhtiyarov, DuPont Pharmaceuticals Co., Experimental Station E400/3410, Wilmington, DE 19880, USA
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