Biochimie (1991) 73, 971-981 © Soci6t6 frangaise de biochimie et biologic mol6culaire / Elsevier, Pads
971
Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers J W e l l e r , W E Hill Division of Biological Sciences. University of Montana, Missoula, MT 59812, USA
(Received 22 October 1990; accepted 4 February 1991)
Summary - - Interactions between Escherichia coil 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to eDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of eDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNA abe. Only the binding of eDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAphe or tRNAfMet alone. The poly(AGU)/tRNA fMet ligand pair caused an additional decline in the binding of eDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the eDNA 'oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for eDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other iigands, caused a large decrease in the binding of the eDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [ 11. rRNA / eDNA / probes / oligomer / structure / function / ribosome / initiation
Introduction
During the initiation of protein biosynthesis the 30S subunit of the ribosome recognizes mRNA, tRNA fM~t, and the three initiation factors, before combining with the 50S subunit to form a complex capable of enzymatically synthesizing proteins [2]. It has been shown that certain regions of the 16S rRNA axe important to the process of initiation. Nucleotides 1533-!,542 caa interact with the Shine-Dalgarno region of m R N A [3-8]. Nucleotide C~400is spatially very close to tRNA in the ribosomal P-site in 30S subunits [9, 10] and the adjacent nucleotides are essential for 30S assembly and function [ 11, 12]. A third region referred to as the 530 loop (nucleotides 518-533), contains a cluster of bases (529-531) which are protected from chemical modification upon the binding of poly(U)-directed tRNA phe [ 13]. The fact that this loop is close to r-protein $3 which has been cross-linked to m R N A [14] and is less reactive to chemical agents in polysomes as compared to free 70S ribosomes [15] provides additional evidence that the loop may be involved in mRNA binding.
All three 16S rRNA sites are phylogenetically highly conserved and all three have a considerable degree of single-stranded character, both in naked 16S rRNA and when the 16S rRNA is incorporated into the 30S subunit [16]. This single-stranded feature presents an obvious mechanism by which rRNA might facilitate ribosomal functions involving other polynucleotides. In previous work we have demonstrated the ability of several putatively single-stranded regions of the ribosomal RNAs to hybridize the eDNA oligomers of varying length, and the ability of these cDNAs to compete with normal ribosomal ligands for binding to their target sites [17]. The sites investigated included those involved in subunit association [ 18], tRNA binding [10, 19], and an antibiotic binding site [20, 21 ]. In the 30S subunit the three 16S rRNA regions on which this study was executed, 517-534, 1390-1417 and 15301542, were first carefully investigated to determine the specific nucleotides accessible to eDNA oligomers. These nucleotides were 16S rRNA bases 1397-1404 and 1534-1542 (Weller and Hill, manuscript in preparation) and 517-528 [22].
972
J Weller, WE Hill
An interesting feature of these three regions is the presence of a pattern, inherent in their nucleotide sequences, which makes them attractive candidates for interacting with mRNAs. This pattern, as described by Trifonov [ l], comprises nucleotides in the repeating order (N-N-G)n, where n _> 3. This complements a sequence found in all procaryotic mRNAs [1, 23]. Trifonov, in addition to noting the occurrence of the pattern, suggested as its function the prevention Of mRNA slippage from its proper reading frame, which results in the synthesis of a faulty protein product. As a test of this hypothesis, we proceeded to determine whether it was possible to detect binding competition between cDNAs to these sites and some of the factors involved in initiation, such as poly(AUG)/tRNA fuet, the initiation factors, and 50S subunits. Because assay ligands are frequently used but do not always behave as the normal components, poly(U) and tRNAPhe were also employed as competitors. The region surrounding the 16S rRNA nucleotide C~40o is sometimes refered to as the 'decoding site', since several independent lines of evidence lead to the conclusion that nucleotide C,40o is in close proximity to the anticodon of tRNA when the ribosome has been programmed with mRNA. The most striking is the crosslink obtained between the 5' wobble baze of the anticodon fo tRNAVak in the P-site, and C~40o of 16S rRNA [9, 24]. Mutagenesis of the nucleotide and its nearest neighbors has shown that the ribosome is extremely labile to changes in sequence in this region [! 2, 25-27]. Electron microscopy has shown the site to be in the cleft of the 30S subunit, and has shown that mRNA-directed tRNAs also appear to be very close to the cleft [28]. Hill and Tassanakajohn [10] showed that a competition could be established between a P-site bound tRNA and a cDNA oligomer lying across C,40o. The region contains nucleotides which change in chemical reactivity upon poly(U)dependent tRNAPhc binding (nucleotide 1408), poly(U)independent tRNAPhe binding (nucleotides 926, 1381, 1399, 1400 and 1401) [13] and subunit association (nucleotide 1394) [29]. We present evidence that the initiation factors alone can cause the displacement of c D N A from 16S rRNA sequences 1397-1404 and from 517-528. Neither initiation factors nor tRNA cause the displacement of cDNA 1534-1541, although, as seen by many others, mRNAs do displace the DNA oligomer from 16S rRNA nucleotides 1534--1541. Since oligomeric cDNAs to the 1400 and 520 regions of 16S rRNA are displaced from the 30S subunit upon the binding of initiation factors and do not bind at all to 70S ribosomes, it seems unlikely that these sites are directly involved in m R N A binding, as suggested by Trifonov. The importance and reactivity of the 1534-1542 antiShine-Dalgarno sequence is supported by our results.
Materials and methods Ribosomes and subunits
Ribosomes were prepared from E coli strain MRE600 (Grain Processing Co, Muscatine, IA), by the methods of Hill et al [30]. Ribosomal subunits were then purified using zonal centrifugation as outlined by Tam and Hill [31 ] except that the subunits were pelleted out of the sucrose by centrifugation rather than by ethanol precipitation. Ribosomes and ribosomal subunits were stored in small aliquots at -70°C. Tight-couple ribosomes (TC 70S) were separated from crude ribosomes by the method of NoU et al [32], as modified by Hill and Tassanakajohn [ 10]. The quality of the subunits and rRNA was monitored as follows: the integrity, of the rRNA was demonstrated by gel electrophoresis, and the subunits were assayed for homogeneity by sedimentation-velocity centrifugation using schlieren optics in a Spinco Model E ultracentrifuge. Functionality was measured by poly(U)-directed tRNAahe binding [33] and the ability to synthesize protein [34]. The specificity of binding of each cDNA oligomer to its target site had been previously demonstrated using RNase H to generate fragments which were displayed upon polyacrylamide gels (Tapprich and Hill, [ 18]). Synthesis and purification of oligodeoxyribonucleotides
Oligodeoxyribonucleotides complementary to regions of 16S rRNA were synthesized on Biosearch model 8600 Automated DNA Synthesizer utilizing J3--cyanoethyl diisopropyll phosphoramidite chemistry. All reagents were obtained from Biosearch with the exception of HPLC grade acetonitrile (Baker) and methylene chloride (Baker). The cDNA oligomers were deblocked according to the manufacturers protocol and purified both before and after the removal of the 5' dimethoxytrityl (DMT) blocking group by reverse-phase high performance liquid chromatography (RP-HPLC) (Gilson). A Column Engineering, C-18 ODS, 5 pm, 25 cm column was employed. Labelling of the cDNA oligomers
Purified DNA oligomers were labelled at the 5'-terminus using [y.32p] ATP (New England Nuclear) and T4 polynucleotyde kinase (United States Biochemical) according to the method of Chaconas and Van de Sande [35]. The cDNA oligomers were purified away from unincorporated nucleotides and salts using Nensorb-20 columns (DuPont) according to the suppliers instructions. Labelling of tRNA and mRNAs
Deacylated tRNAfMet was 5'-labelled with p32 according to the method of Lill and Wintermeyer [36]. Deacylated tRNA gives the same protection patterns on 30S subunits as charged tRNAs [13], binds with the same kinetics [37] and is more easily prepared as an homogeneous species. MS2 mRNA and poly(AGU) (Pharmacia) were first dephosphorylated with CIAP (calf intestinal alkaline phosphatase from Bethesda Research Labs) according to the suppliers instructions, precipitated with 2 vol of ethanol and then resuspended in buffer to allow labelling with T4 polynucleotide kinase as described above. Buffers
Assays and competitions were caried out in IX binding buffer: 150 mM KCI or NH4CI2, 15 mM MgCl2, 20 mM Tris-HCl pH 7.4, 0.2 M DTT, 0.5 mM GTP.
Probing ribosome initiation complex
Initiation factors Crude initiation factors were prepared as described in Traub et ai [34]. The crude initiation factor preparation was assayed in a tRNA-binding assay using poly(AGU)-directed tRNA fMet, and was found to give maximum efficiency when 10 lal was used with 15 pmol of ribosomes, so this amount was used for all subsequent assays. We were able to assay a pure initiation factor three (iF3) preparation thanks to the kind gift of E Wickstrom. IF3 was introduced into the reactions at a ratio of 4:1 mol/mol 30S subunit.
cDNA oligomer binding and specificity of binding The level and site specificity of eDNA oligomer binding to the 30S ribosomal subunit or 70S ribosome was established in previous experiments (517-528, Camp, [22]; 1398-1403, unpublished data; 1534-1541, unpublished data).
Filter binding assays The 30S-oligonucleotide complex, or the 70S-oligonucleotide complex, was assayed for changes in the cDNA binding level caused by the presence of the other iigands using nitrocellulose filters (0.45 lam HAWP, Millipore). After ilicubation of the indicated species, the assay mixtures were diluted to ! ml with 1X binding buffer (which did not contain the DTT or GTP) and immediately filtered through pre-soaked 0.45 lam HAWP filters. Two l-ml rinses with the same buffer followed. The filters were immersed in Scintiverse scintillation cocktail (Fisher Biochemical), and the isotopic emissions were detected in a Packard scintillation counter.
973
3) Oligonucleotide vs initiation factors: crude initiation factors (elF) were added to the reactions at 10 lal per reaction. Additional reactions were run using 5 lag of poly(AGU) per reaction and 45 pmol of tRNA in addition to the crude IF. Similar reactions were done using 60 pmol IF3 per reaction in place of the clF fraction.
Sucrose gradient co-migration assays on reassociated subunits One hundred pmol 30S subunits were incubated with a 20:1 excess of the indicated eDNA oligomer having a specific activity of about 1000 cpm/pmol, in a vol of 50 lal of 1X binding barfer. After a 30 min incubation period at 37°C, 90 pmol of activated 50S subunits in 50 lal of IX binding buffer were added, and the reaction was incubated for a further 30 rain before being layered onto a 5 ml 5-26% linear sucrose gradient (in IX binding buffer). The gradients were centrifuged in a Beckman SW50. I rotor at 45 000 rpm (300 000 g) for 2.5 h. The gradients were dripped into 15 equal fractions and both the A2t~ and the cpm/lal of each fraction were monitored. Each experiment was performed in duplicate, and each experiment with a given eDNA oligomer was performed 2 separate times.
Results Complementary oligodeoxyribonucleotide synthesis
Competition assays Simultaneous addition of ligands and cDNA In these experiments the cDNA oligomer and the competing ligand(s) were added simultaneously, incubated for 4 h and then processed as described above. The experiments were carried out with either the oligonucleotide labelled or with the other ligand labelled. All experiments were performed in triplicate, and each experiment was performed a minimum of 2 different times.
T h e s e c o n d a r y structure m a p proposed b y N o l l e r et al [38], was used to d e s i g n the sequence o f several oligod e o x y r i b n o n u c l e o t i d e s w h i c h w o u l d c o m p l e m e n t the d e s i r e d target sites, as s h o w n in figure 1. All o f the target sites are p r o p o s e d to be single-stranded. In the sections w h i c h follow, the e D N A o l i g o m e r s are desig n a t e d a c c o r d i n g to the 16S r R N A s e q u e n c e to which they are c o m p l e m e n t a r y .
Displacement reactions In this series of experiments the labelled ligand (cDNA or its competitor) was pre-incubated with the 30S subunits for 30 min, the 2nd reactant was added, the whole reaction was then incubated for 4 h and processed as described above.
Reaction mixtures In all reactions 15 pmol of 30S subunits or 70S ribosomes were used, and 300 pmol of the oligonucleotide were tested. For the tRNA binding assays 5 lag poly(AGU) or poly(U) per reaction were used. A total vol of 30 lal was used.
Reaction mixtures assayed 1) Oligonucleotide vs RNA:tRNA Phe or tRNA fMet was added to the reactions at ratios of 0, 1, 2, 3:1 mol/mol. When programming the ribosomes the same ratios were used but poly(U) or poly(AGU) was present in all reactions at 5 lag per reaction, and for comparison some reactions were made with 15 pmol of MS2 RNA. 2) Oligonucleotide vs poly(AGU) or poly(U): poly (AGU) or poly(U) was added to the reactions at a saturing level (0.15 A26o or 5 lag), to determine whether there was an effect upon or by cDNA oligomer binding. For comparison several reactions were run with 15 pmol of MS2 RNA.
Hybridization o f oligodeoxyribonucleotides to 30S ribosomal subunits and to 70S ribosomes T h e p e r c e n t a g e o f 30S subunits or tight-couple 70S r i b o s o m e s w h i c h f o r m e d c o m p l e x e s with the e D N A o l i g o m e r s was a s s a y e d b y nitrocellulose m e m b r a n e filtration. T a b l e s I a n d II list the m a x i m a l percentage o f b i n d i n g o b s e r v e d . F i g u r e 2 s h o w s s o m e saturation b i n d i n g curves o f e D N A oligomers. T h e b i n d i n g o f the c D N A s to 30S s u b u n i t s and, in .subunit r e a s s o c i a t i o n assays, to 70S r i b o s o m e s was also a s s a y e d u s i n g sucrose gradient c o m i g r a t i o n . The results are s h o w n in figures 3 and 4. The e D N A 1 5 3 4 - 1 5 4 1 b o u n d e q u a l l y well to 70S r i b o s o m e s and to 30S subunits. T h e e D N A 1 3 9 6 - 1 4 0 3 did not bind to r e - f o r m e d 70S r i b o s o m e s and T C 70 r i b o s o m e s , but did not d e c r e a s e the a m o u n t o f subunit association. S i m i l a r results w e r e obtained for the e D N A 5 1 7 - 5 2 8 [22].
974
J Weller, WE Hill
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971
Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers J W e l l e r , W E Hill Division of Biological Sciences. University of Montana, Missoula, MT 59812, USA
(Received 22 October 1990; accepted 4 February 1991)
Summary - - Interactions between Escherichia coil 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to eDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of eDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNA abe. Only the binding of eDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAphe or tRNAfMet alone. The poly(AGU)/tRNA fMet ligand pair caused an additional decline in the binding of eDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the eDNA 'oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for eDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other iigands, caused a large decrease in the binding of the eDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [ 11. rRNA / eDNA / probes / oligomer / structure / function / ribosome / initiation
Introduction
During the initiation of protein biosynthesis the 30S subunit of the ribosome recognizes mRNA, tRNA fM~t, and the three initiation factors, before combining with the 50S subunit to form a complex capable of enzymatically synthesizing proteins [2]. It has been shown that certain regions of the 16S rRNA axe important to the process of initiation. Nucleotides 1533-!,542 caa interact with the Shine-Dalgarno region of m R N A [3-8]. Nucleotide C~400is spatially very close to tRNA in the ribosomal P-site in 30S subunits [9, 10] and the adjacent nucleotides are essential for 30S assembly and function [ 11, 12]. A third region referred to as the 530 loop (nucleotides 518-533), contains a cluster of bases (529-531) which are protected from chemical modification upon the binding of poly(U)-directed tRNA phe [ 13]. The fact that this loop is close to r-protein $3 which has been cross-linked to m R N A [14] and is less reactive to chemical agents in polysomes as compared to free 70S ribosomes [15] provides additional evidence that the loop may be involved in mRNA binding.
All three 16S rRNA sites are phylogenetically highly conserved and all three have a considerable degree of single-stranded character, both in naked 16S rRNA and when the 16S rRNA is incorporated into the 30S subunit [16]. This single-stranded feature presents an obvious mechanism by which rRNA might facilitate ribosomal functions involving other polynucleotides. In previous work we have demonstrated the ability of several putatively single-stranded regions of the ribosomal RNAs to hybridize the eDNA oligomers of varying length, and the ability of these cDNAs to compete with normal ribosomal ligands for binding to their target sites [17]. The sites investigated included those involved in subunit association [ 18], tRNA binding [10, 19], and an antibiotic binding site [20, 21 ]. In the 30S subunit the three 16S rRNA regions on which this study was executed, 517-534, 1390-1417 and 15301542, were first carefully investigated to determine the specific nucleotides accessible to eDNA oligomers. These nucleotides were 16S rRNA bases 1397-1404 and 1534-1542 (Weller and Hill, manuscript in preparation) and 517-528 [22].
972
J Weller, WE Hill
An interesting feature of these three regions is the presence of a pattern, inherent in their nucleotide sequences, which makes them attractive candidates for interacting with mRNAs. This pattern, as described by Trifonov [ l], comprises nucleotides in the repeating order (N-N-G)n, where n _> 3. This complements a sequence found in all procaryotic mRNAs [1, 23]. Trifonov, in addition to noting the occurrence of the pattern, suggested as its function the prevention Of mRNA slippage from its proper reading frame, which results in the synthesis of a faulty protein product. As a test of this hypothesis, we proceeded to determine whether it was possible to detect binding competition between cDNAs to these sites and some of the factors involved in initiation, such as poly(AUG)/tRNA fuet, the initiation factors, and 50S subunits. Because assay ligands are frequently used but do not always behave as the normal components, poly(U) and tRNAPhe were also employed as competitors. The region surrounding the 16S rRNA nucleotide C~40o is sometimes refered to as the 'decoding site', since several independent lines of evidence lead to the conclusion that nucleotide C,40o is in close proximity to the anticodon of tRNA when the ribosome has been programmed with mRNA. The most striking is the crosslink obtained between the 5' wobble baze of the anticodon fo tRNAVak in the P-site, and C~40o of 16S rRNA [9, 24]. Mutagenesis of the nucleotide and its nearest neighbors has shown that the ribosome is extremely labile to changes in sequence in this region [! 2, 25-27]. Electron microscopy has shown the site to be in the cleft of the 30S subunit, and has shown that mRNA-directed tRNAs also appear to be very close to the cleft [28]. Hill and Tassanakajohn [10] showed that a competition could be established between a P-site bound tRNA and a cDNA oligomer lying across C,40o. The region contains nucleotides which change in chemical reactivity upon poly(U)dependent tRNAPhc binding (nucleotide 1408), poly(U)independent tRNAPhe binding (nucleotides 926, 1381, 1399, 1400 and 1401) [13] and subunit association (nucleotide 1394) [29]. We present evidence that the initiation factors alone can cause the displacement of c D N A from 16S rRNA sequences 1397-1404 and from 517-528. Neither initiation factors nor tRNA cause the displacement of cDNA 1534-1541, although, as seen by many others, mRNAs do displace the DNA oligomer from 16S rRNA nucleotides 1534--1541. Since oligomeric cDNAs to the 1400 and 520 regions of 16S rRNA are displaced from the 30S subunit upon the binding of initiation factors and do not bind at all to 70S ribosomes, it seems unlikely that these sites are directly involved in m R N A binding, as suggested by Trifonov. The importance and reactivity of the 1534-1542 antiShine-Dalgarno sequence is supported by our results.
Materials and methods Ribosomes and subunits
Ribosomes were prepared from E coli strain MRE600 (Grain Processing Co, Muscatine, IA), by the methods of Hill et al [30]. Ribosomal subunits were then purified using zonal centrifugation as outlined by Tam and Hill [31 ] except that the subunits were pelleted out of the sucrose by centrifugation rather than by ethanol precipitation. Ribosomes and ribosomal subunits were stored in small aliquots at -70°C. Tight-couple ribosomes (TC 70S) were separated from crude ribosomes by the method of NoU et al [32], as modified by Hill and Tassanakajohn [ 10]. The quality of the subunits and rRNA was monitored as follows: the integrity, of the rRNA was demonstrated by gel electrophoresis, and the subunits were assayed for homogeneity by sedimentation-velocity centrifugation using schlieren optics in a Spinco Model E ultracentrifuge. Functionality was measured by poly(U)-directed tRNAahe binding [33] and the ability to synthesize protein [34]. The specificity of binding of each cDNA oligomer to its target site had been previously demonstrated using RNase H to generate fragments which were displayed upon polyacrylamide gels (Tapprich and Hill, [ 18]). Synthesis and purification of oligodeoxyribonucleotides
Oligodeoxyribonucleotides complementary to regions of 16S rRNA were synthesized on Biosearch model 8600 Automated DNA Synthesizer utilizing J3--cyanoethyl diisopropyll phosphoramidite chemistry. All reagents were obtained from Biosearch with the exception of HPLC grade acetonitrile (Baker) and methylene chloride (Baker). The cDNA oligomers were deblocked according to the manufacturers protocol and purified both before and after the removal of the 5' dimethoxytrityl (DMT) blocking group by reverse-phase high performance liquid chromatography (RP-HPLC) (Gilson). A Column Engineering, C-18 ODS, 5 pm, 25 cm column was employed. Labelling of the cDNA oligomers
Purified DNA oligomers were labelled at the 5'-terminus using [y.32p] ATP (New England Nuclear) and T4 polynucleotyde kinase (United States Biochemical) according to the method of Chaconas and Van de Sande [35]. The cDNA oligomers were purified away from unincorporated nucleotides and salts using Nensorb-20 columns (DuPont) according to the suppliers instructions. Labelling of tRNA and mRNAs
Deacylated tRNAfMet was 5'-labelled with p32 according to the method of Lill and Wintermeyer [36]. Deacylated tRNA gives the same protection patterns on 30S subunits as charged tRNAs [13], binds with the same kinetics [37] and is more easily prepared as an homogeneous species. MS2 mRNA and poly(AGU) (Pharmacia) were first dephosphorylated with CIAP (calf intestinal alkaline phosphatase from Bethesda Research Labs) according to the suppliers instructions, precipitated with 2 vol of ethanol and then resuspended in buffer to allow labelling with T4 polynucleotide kinase as described above. Buffers
Assays and competitions were caried out in IX binding buffer: 150 mM KCI or NH4CI2, 15 mM MgCl2, 20 mM Tris-HCl pH 7.4, 0.2 M DTT, 0.5 mM GTP.
Probing ribosome initiation complex
Initiation factors Crude initiation factors were prepared as described in Traub et ai [34]. The crude initiation factor preparation was assayed in a tRNA-binding assay using poly(AGU)-directed tRNA fMet, and was found to give maximum efficiency when 10 lal was used with 15 pmol of ribosomes, so this amount was used for all subsequent assays. We were able to assay a pure initiation factor three (iF3) preparation thanks to the kind gift of E Wickstrom. IF3 was introduced into the reactions at a ratio of 4:1 mol/mol 30S subunit.
cDNA oligomer binding and specificity of binding The level and site specificity of eDNA oligomer binding to the 30S ribosomal subunit or 70S ribosome was established in previous experiments (517-528, Camp, [22]; 1398-1403, unpublished data; 1534-1541, unpublished data).
Filter binding assays The 30S-oligonucleotide complex, or the 70S-oligonucleotide complex, was assayed for changes in the cDNA binding level caused by the presence of the other iigands using nitrocellulose filters (0.45 lam HAWP, Millipore). After ilicubation of the indicated species, the assay mixtures were diluted to ! ml with 1X binding buffer (which did not contain the DTT or GTP) and immediately filtered through pre-soaked 0.45 lam HAWP filters. Two l-ml rinses with the same buffer followed. The filters were immersed in Scintiverse scintillation cocktail (Fisher Biochemical), and the isotopic emissions were detected in a Packard scintillation counter.
973
3) Oligonucleotide vs initiation factors: crude initiation factors (elF) were added to the reactions at 10 lal per reaction. Additional reactions were run using 5 lag of poly(AGU) per reaction and 45 pmol of tRNA in addition to the crude IF. Similar reactions were done using 60 pmol IF3 per reaction in place of the clF fraction.
Sucrose gradient co-migration assays on reassociated subunits One hundred pmol 30S subunits were incubated with a 20:1 excess of the indicated eDNA oligomer having a specific activity of about 1000 cpm/pmol, in a vol of 50 lal of 1X binding barfer. After a 30 min incubation period at 37°C, 90 pmol of activated 50S subunits in 50 lal of IX binding buffer were added, and the reaction was incubated for a further 30 rain before being layered onto a 5 ml 5-26% linear sucrose gradient (in IX binding buffer). The gradients were centrifuged in a Beckman SW50. I rotor at 45 000 rpm (300 000 g) for 2.5 h. The gradients were dripped into 15 equal fractions and both the A2t~ and the cpm/lal of each fraction were monitored. Each experiment was performed in duplicate, and each experiment with a given eDNA oligomer was performed 2 separate times.
Results Complementary oligodeoxyribonucleotide synthesis
Competition assays Simultaneous addition of ligands and cDNA In these experiments the cDNA oligomer and the competing ligand(s) were added simultaneously, incubated for 4 h and then processed as described above. The experiments were carried out with either the oligonucleotide labelled or with the other ligand labelled. All experiments were performed in triplicate, and each experiment was performed a minimum of 2 different times.
T h e s e c o n d a r y structure m a p proposed b y N o l l e r et al [38], was used to d e s i g n the sequence o f several oligod e o x y r i b n o n u c l e o t i d e s w h i c h w o u l d c o m p l e m e n t the d e s i r e d target sites, as s h o w n in figure 1. All o f the target sites are p r o p o s e d to be single-stranded. In the sections w h i c h follow, the e D N A o l i g o m e r s are desig n a t e d a c c o r d i n g to the 16S r R N A s e q u e n c e to which they are c o m p l e m e n t a r y .
Displacement reactions In this series of experiments the labelled ligand (cDNA or its competitor) was pre-incubated with the 30S subunits for 30 min, the 2nd reactant was added, the whole reaction was then incubated for 4 h and processed as described above.
Reaction mixtures In all reactions 15 pmol of 30S subunits or 70S ribosomes were used, and 300 pmol of the oligonucleotide were tested. For the tRNA binding assays 5 lag poly(AGU) or poly(U) per reaction were used. A total vol of 30 lal was used.
Reaction mixtures assayed 1) Oligonucleotide vs RNA:tRNA Phe or tRNA fMet was added to the reactions at ratios of 0, 1, 2, 3:1 mol/mol. When programming the ribosomes the same ratios were used but poly(U) or poly(AGU) was present in all reactions at 5 lag per reaction, and for comparison some reactions were made with 15 pmol of MS2 RNA. 2) Oligonucleotide vs poly(AGU) or poly(U): poly (AGU) or poly(U) was added to the reactions at a saturing level (0.15 A26o or 5 lag), to determine whether there was an effect upon or by cDNA oligomer binding. For comparison several reactions were run with 15 pmol of MS2 RNA.
Hybridization o f oligodeoxyribonucleotides to 30S ribosomal subunits and to 70S ribosomes T h e p e r c e n t a g e o f 30S subunits or tight-couple 70S r i b o s o m e s w h i c h f o r m e d c o m p l e x e s with the e D N A o l i g o m e r s was a s s a y e d b y nitrocellulose m e m b r a n e filtration. T a b l e s I a n d II list the m a x i m a l percentage o f b i n d i n g o b s e r v e d . F i g u r e 2 s h o w s s o m e saturation b i n d i n g curves o f e D N A oligomers. T h e b i n d i n g o f the c D N A s to 30S s u b u n i t s and, in .subunit r e a s s o c i a t i o n assays, to 70S r i b o s o m e s was also a s s a y e d u s i n g sucrose gradient c o m i g r a t i o n . The results are s h o w n in figures 3 and 4. The e D N A 1 5 3 4 - 1 5 4 1 b o u n d e q u a l l y well to 70S r i b o s o m e s and to 30S subunits. T h e e D N A 1 3 9 6 - 1 4 0 3 did not bind to r e - f o r m e d 70S r i b o s o m e s and T C 70 r i b o s o m e s , but did not d e c r e a s e the a m o u n t o f subunit association. S i m i l a r results w e r e obtained for the e D N A 5 1 7 - 5 2 8 [22].
974
J Weller, WE Hill
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