212

Bloc&mica et Biopt~vsica Acta, 1087 (1990) 212 218 Elsevier

BBAEXP 92176

Nuclease $1 mapping of 16S ribosomal R N A in ribosomes Mohammad A. Rahman* and Henry W. Schaup Department of Biochemist O, and Biophysics, Oregon State UniversiO,, Corvallis, OR ( U. S. A.) (Received 18 June 1990)

Key words: rRNA; RNA, 16S; Ribosome; Nuclease S1 ; ( E. coil )

Escherichia coil 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which

are believed to be single-stranded in ribosomes. The quantitative disposition of these sequences within ribosomes is not known. Here we describe experiments, designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes. Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired. Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA. The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1. In deproteinated E. coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs. However, the quantitative disposition of the sequences in 70S ribosomes varies with each position. In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated.

Introduction Ribosomes, particles composed of proteins and RNA~ play a prominent part in the synthesis of proteins. Concise roles for R N A elements in ribosomes have not been established. Highly conserved single-stranded regions of 16S rRNA and 16S-like rRNAs in 30S ribosomal subunits offer simple structural elements, which may be envisioned to participate in protein synthesis through base-pairing interactions with non-ribosome components [1,2]. Among the universally conserved single-stranded sequences, the regions 517-533, 13941408 and 1492-1506 have the longest single-stranded stretches [3-6]. The 3' terminal sequence C C U C C (position 1535-1539) is universally present in eubacteria and archaebacteria [3,5,6], with one exception [7], but not in eukaryotes. The remarkable conservation of the single-stranded portions of 16S-like rRNAs implies that they must be active in some global function in protein synthesis. For example, the conserved eubacterial 3' terminal sequence C C U C C is clearly essential for ini-

* Present address: Roche Institute of Molecular Biology, Nutley, NJ 07110, U.S.A. Correspondence: H.W. Schaup, Dept. Biochem. Biophys., Oregon State University, Corvallis, OR 97331, U.S.A.

tiation of protein synthesis [8-13]. The zone centered about C1400 in 16S rRNA has been implicated in tRNA binding [14-18]. A knowledge of rRNA sequence deportment is necessary to propose and support theories about the function of conserved portions of rRNA in ribosomes and ribosomal subunits. There are current models showing the three-dimensional arrangement of E. coli 16S rRNA in 30S subunits [19 22]. Support for these representations comes from various techniques such as comparative sequence analysis, chemical and enzymatic footprinting, R N A - R N A and RNA-protein crosslinking and neutron diffraction studies. However, these techniques do not readily permit determination of the number of ribosomes in a given configuration. We have used short site-specific DNA oligomers to infer the single strandedness of conserved portions of 16S rRNA in 30S subunits and 70S ribosomes from E. coll. Unlike previous work by others~ the probing with the D N A should not cause artifactual alterations in the particle being investigated. The experiments were undertaken using conditions which maintain ribosome configuration. We have found that the regions centered about positions 525, 1400, 1500 and 1535 are quantitatively singlestranded in 16S r R N A and nearly so in 30S subunits. In active 70S ribosomes, sequences centered about positions 520 and 1400 are open in roughly one-half of the ribosomes. The sequence zones centered about positions

0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B,V. (Biomedical Division)

213 1500 and 1538 are only open in about 30% of the ribosomes. We have also found that the presence of DNA probes complementary to other highly conserved single-stranded zones does not alter the interactions observed when the D N A probes are tested individually. Materials and Methods

Preparation of 70S ribosomes, 30S subunits and 16S rRNA The 70S ribosomes, 30S subunits and 16S rRNAs were prepared from E. coli D-10 cells harvested in the early log phase of growth. The cells were stored frozen at - 7 0 ° C . Ribosomes and ribosomal subunits were prepared by zone ultracentrifugation using previously described salt washing procedures [23,24]. To make 70S and 30S particles, cells were suspended in TSM (10 mM Tris/3 mM succinic acid/10 mM MgC12/6 mM 2 mercaptoethanol (pH 8)) then passed through a French pressure cell at 15 000 psi [21]. The lysate was cleared of cell debris by low speed centrifugation (15 000 × g for 10 rain). Ammonium sulfate was added (210 mg/ml) to the supernate to further clarify the lysate. Following centrifugation at 15000 X g and an additional 210 m g / m l of sulfate was added to precipitate 70S particles which were resuspended in TSM and made to 0.6 M NHaSO 4. The 70S particles were then pelleted by ultracentrifugation (150000 × g for 3 h). Pellets were resuspended in TSM and dialyzed against TSM prior to storage or use. Particles and subunits prepared in this way are active in in vitro protein synthesis. In vitro translations were done using MS2 m R N A and purified factors as described previously [9]. The particles were judged to be active on the basis of incorporation of radio labeled amino acids into MS2 coat protein as previously reported [9]. These procedures yield 70S particles which are associated with m R N A and tRNAs. Ribosomal RNA was prepared by zone ultracentrifugation from phenol extracts of cell lysates cleared of cell debris as described elsewhere [25,26]. Synthesis and labeling of DNA oligomer probes DNA oligomer probes (Table I) were chemically synthesized and supplied by the Central Service Laboratory of the Center for Gene Research and Biotechnology, Oregon State University. The probes were made with an Applied BioSystems Model 380A DNA Synthesizer using phosphoramidite chemistry [27,28]. HPLC purified probes were 5' end labeled with [y-32p]ATP (adenosine 5'-triphosphate [y_32p], specific activity 7000 mCi/mmol, crude; ICN Radiochemicals) and polynucleotide kinase (BRL) according to established methods [29-31]. The kinated probes were purified by 20% polyacrylamide-7 M urea gel electrophoresis. After autoradiography the zone containing the full length 5"

end-labeled probe was cut out and the DNA was eluted from the gel essentially as previously described [31].

3' end labeling of 16S rRNA 16S rRNA was 3' end labeled by the method described by England et al. [32], using [5'-32p]pCp (cytidine 3'-5' bis(phosphate), (New England Nuclear) specific activity 3000 Ci/mmol) and T4 RNA ligase (BRL) incubated overnight at 4°C in ligase buffer (50 mM Hepes/20 m M MGC12/3.3 mM DTT/0.015 ~g~1-1 BSA). The labeled RNA was purified using a 2.8% polyacrylamide 7 M urea gel electrophoresis system and was recovered from the gel by two extractions with equal volumes of SSC buffer (150 mM NaC1/15 mM sodium citrate (pH 7.2)) and redistilled phenol (equilibrated with SSC buffer) [33], followed by ethanol precipitation at - 20°C. The suspension was then passed through a C F l l (Cellulose Powder, Whatman) column prepared in a 1 ml pipet tip plugged with silanized glass wool. The column was washed with 5 ml of 80% ethanol and then with 5 ml of 80% methanol. This process removed acrylamide. The pCp-labeled 16S rRNA was eluted from the C F l l column with 500 /~1 of sterile distilled water, lypholized and dissolved in 20 /L1 of TSM buffer and the specific activity was determined. RNA-DNA hybridization and RNase H cleavage 3' end-labeled 16S rRNA was mixed with a 30-fold molar excess of unlabeled DNA probes in 10/~1 of E. coli RNase H hybridization buffer (40 mM Tris-base (pH 7.9)/4 mM MgC12/1 mM dithiothreitol/0.03 btg /d -1 BSA) and calf thymus RNase H hybridization buffer (50 mM Tris-base (pH 8.3)/25 mM MgC12/0.1 M KC1/0.5 txg'/~1-1 BSA), heated at 50°C for 20 min, then cooled to 32°C and further incubated at 32°C for 30 rain [34]. After addition of RNase H (EC 3.1.26.4; BRL) to a concentration of 0.5 U//Lg of 16S rRNA, the reactions were incubated at 32°C for 50 min. 20 /~1 of tracking dye (10 M urea/0.05% xylene cyanol/0.05% bromphenol blue/5 mM Tris-borate/1.0 mM EDTA (pH 8.3)) [35] were added to the reactions which were then analyzed electrophoretically on 2.8% polyacrylamide-7 M urea gels. Hybridization of DN,4 probes to 16S rRNA, 30S subunits, 70S ribosomes and digestion with nuclease S1 Hybridization of DNA probes to 16S rRNA, 30S subunits and 70S ribosomes in nuclease S1 protection experiments was carried out with minor modifications of described procedures [36-39]. The experimental samples were incubated with a 15-fold molar excess of DNA probe in 20 #1 of S1 hybridization buffer modified by the addition of MgC12 to stabilize the configuration of the ribosomes and the RNA (40 mM Pipes/400 m M NaC1/3 mM MgC12 (pH 6.8)). The incubations were done at 37°C for 30 min, slowly

214 TABLE I

T A B L E II

Synthetic D N A s a

Potential hybrids between D N A 520 and 16S r R N A

DNA

Position b

Length

Sequence ~

520 l 396 1475 1 493 1 533

5 2 0 - 530 1396-1404 1 475-1485 1 493-1504 1 533-1542

11 9 11 12 10

TCGTCGGCGCC ACACCGCCC CTAAGTACTGA TCAGCATTGTTC GTGEIAGGAAT

a Position a n d sequence are referenced to E. coli 16S r R N A . b Position s p a n n e d on 16S r R N A . c Sequences are s h o w n in the 3' to 5' direction.

Position a 98-

Hybrid b

108

Position ~

ACGAGUGGCGG

350-

Hybrid b

360

TCGTCGGCGCC 435

445

TCGTCGGCGCC

ACUUUCAGCGG

520

530

TCGTCGGCGCC 681

691

:

932- 942

:'-':

935- 945

::.',

TCGTCGGCGCC

ACAAGCGGUGG

1 346-1 356

TCGTCGGCGCC 1 516-1 526

CGCACAAGCGG "::

TCGTCGGCGCC

cooled to 30°C and the incubation was continued at 30°C for 3 h. Nuclease S1 (EC 3.1.4.-, Pharmacia) was diluted to a concentration of 5 U / ~ l with nuclease $1 digestion buffer prior to use and kept at 0°C (30 mM sodium acetate/280 mM NaC1/4.5 mM zinc sulfate/5% glycerol (v/v) (pH 4.6)). 30 ~1 of S1 digestion buffer was added to each reaction followed by incubation at 30°C for 45 rain. The reaction was stopped by the addition of 25/xl of termination mix (2.5 M ammonium acetate/50 mM EDTA) as well as 25 /~g of tRNA as carrier [38]. The mixture was then precipitated by adding 2.5 vol of ethanol. The precipitates were dissolved in 25 ~tl of loading buffer (95 mM Tris-borate (pH 8.3)/1.0 mM E D T A / 7 M urea/0.01% xylene c y a n o l / 0.01% bromphenol blue) [35] and analyzed by 20% acrylamide-7 M urea gel electrophoresis.

AGCAGCCGCGG TCGTCGGCGCC

AGGUGUAGCGG "_-

GGCAGCAGUGG

AGUAAUCGUGG TCGTCGGCGCC

GGAACCUGCGG TCGTCGGCGCC

Position s p a n n e d on 16S r R N A . h r R N A is s h o w n in the upper sequence in 5' to 3' orientation and D N A 520 is represented in the lower sequence.

commonly used for S1 nuclease digestions contain no magnesium and the pH is usually 4.6. Therefore, an assessment of the enzyme's activity under our conditions was necessary. The results of a polyacrylamide gel analysis of nuclease S1 digestions at various enzyme concentrations in the modified buffer are presented in Fig. 1. The autoradiograph in Fig. 2 shows the effect of increasing concentration of D N A probe 520 relative to

Results M

1

2

3

4

5

7.5

10

T

Screening for potential partial hybrids The D N A probes used in these investigations are shown in Table ]. An analysis was made to find all potential points within 16S r R N A where partial hybrids could be formed which could cause ambiguity in interpreting nuclease $1 experiments. For D N A probe 520, Table II shows some potential hybrids of two or more contiguous base pairs with 16S rRNA. When compared with current secondary structure models for 16S rRNA, most of the alignments are in double-stranded or partially double-stranded zones of the molecule. Stable hybrids can not form at these sites under the conditions used in these investigations. The only alignment that would yield full nuclease $1 protection of the D N A is the one to which the D N A probe is fully complementary when the zone is single stranded. Other interactions would yield digestion products very much smaller than the intact probe. This is also true for all the other D N A probes used in these investigations.

DNA binding to 16S rRNA The nuclease S1 hybridization buffer employed here had a pH of 6.8 and contained magnesium. Buffers

Fig. 1. A u t o r a d i o g r a p h showing nuclease S1 activity. 5 picomol of 5'-32P-labeled D N A 520 was digested with various a m o u n t s of nuclease S1. T h e lane n u m b e r s c o r r e s p o n d to the n u m b e r of units per /~1 of the nuclease S1 used in each reaction. Lane M s h o w s intact D N A 520 a n d lane T s h o w s a digestion u s i n g 1 # g / l u l of nuclease S1 in the presence of 3.43.10 -12 mol of yeast t R N A . T h e polyacryla m i d e gel was a 20% d e n a t u r i n g gel ( 2 6 x 2 4 . 8 × 0 . 0 8 cm) run at 25 mA for 150 rain. T h e digestion conditions were as described in Materials and Methods.

215 a fixed level of 16S rRNA. Similar experiments were done using D N A probes 1396, 1493 and 1533. After autoradiography of the gels, the zones containing D N A were cut out and quantitated using a liquid scintillation spectrometer. The molar amount of probe D N A bound to 16S r R N A was calculated using the known specific activity of the D N A probes and plotted against molar excess to generate the saturation hybridization curves. Fig. 3A,C shows such plots using data obtained from experiments with probe 520 and 1396. Fig. 3B,D,E,F show Scatchard plots [40,41] using data for D N A 520, 1396, 1493 and 1533. In all cases n = approx. 1 (where n is the number of binding sites per molecule of 16S RNA). The binding appears to be one-to-one as expected for specific interactions. From Fig. 3A,C it is evident that the binding of the probes to 16S r R N A reaches saturation in the presence of a 15- to 20-fold molar excess of the DNA. We used probe 1475 as a control for nonspecific D N A - r R N A or DNA-ribosomal protein interactions. It was an 11 nucleotide D N A complementary to positions 1475-1485 in E. coli 16S rRNA. The complement to this probe in current secondary structural models of 16S rRNA is shown to be in a conserved double-stranded region of the molecule. Protection of the probe from nuclease S1 digestion would not be expected unless the D N A was involved in secondary artifactual interactions. There was no observable protection from nuclease S1 of probe 1475 after incubation with 16S rRNA, 30S subunits, or with 70S ribosomes (Fig. 4). This experiment shows that hybridization of the probes to 16S rRNA is essential for protection. It also shows that secondary interactions, possibly with ribosomal pro-

M

1

2

5

10

15

20

25

30

Fig. 2. Saturation hybridization of DNA 520 with 16S rRNA. Autoradiograph of a 2055 polyacrylamide gel analysis of nuelease $1 resistant 5 '-32p-labeled DNA 520 hybridized to 1.67.10-13 tool of 16S rRNA. The numbers on top indicate the molar ratio of DNA to 16S rRNA in the reactions. M shows intact DNA 520. The reaction conditions axe as described in Materials and Methods. Uneven migration across the gel is due to different amounts of salts in the samples and an edge effect.

TABLE III

Binding stoichiometry of DNAs complementary to 16S rRNA a DNA

DNA/16S

DNA/30S

DNA/70S

520 1396 1493 1533

0.87 0.99 0.80 1.02

0.69 0.90 0.73 0.73

0.61 0.57 0.24 0.30

a Expressed as molar ratios.

teins, are not likely to produce artifacts in these experiments.

RNase H cleavage pattern To provide further assurance that the probes were interacting with the expected zones, we analyzed RNase H digestion patterns of the 16S r R N A hybridized to our probes. The conditions used were comparable to those adapted for nuclease S1 digestions. RNase H will only cleave R N A / D N A hybrids. Therefore, the size of the digestion products in these experiments is predictable only if the probe has specifically hybridized to the 16S r R N A [34,42,43]. The results of such an experiment with probe 520 are shown in Fig. 5. The 16S r R N A used in these experiments was Y-end labeled. Specific cleavage in the presence of probe 520 would, and did, yield a single 3' end radiolabeled fragment of expected size. Similar experiments with probes 1396, 1493 and 1533 have yielded only one RNase H digestion product and those were also of the expected size (unpublished data). Protection of DNA oligonucleotide probes in the presence of 16S rRNA, 30S subunits and 70S ribosomes The protection of the site-specific D N A probes 1396 and 1493 in the presence of 16S rRNA, 30S subunits and 70S ribosomes is shown in Fig. 6A,B. In these experiments equimolar amounts of ribosomes or components from them were incubated with a 15-fold molar excess of individual D N A probes (5'-end labeled) and then subjected to nuclease $1 attack. After polyacrylamide gel separation and autoradiography the bands were cut out of the gel and quantitated by liquid scintillation spectrometry. The number of moles of D N A bound per mole of 16S rRNA, 30S subunits or 70S ribosomes were calculated from the specific activity of the respective probes. The binding stoichiometry was determined for all of the D N A probes in this way and the results are shown in Table III. These experiments were also done in the presence of unlabeled mixtures of each of the other probes. Each unlabeled probe was present at 20-fold molar excess over the ribosomal component. The results of those experiments were no different from those observed when the probes were present individually. This means that there are no cooperative interactions which would reduce or increase

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Nuclease S1 mapping of 16S ribosomal RNA in ribosomes.

Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded ...
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