©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distribution of Cross-links between mRNA Analogues and 16 S rRNA in Escherichia coli 70 S Ribosomes Made under Equilibrium Conditions and Their Response to tRNA Binding (*)

Dalia I. Juzumiene (§) , Tatjana G. Shapkina , Paul Wollenzien (¶)

From the (1) Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interaction between mRNA and Escherichia coli ribosomes has been studied by photochemical cross-linking using mRNA analogues that contain 4-thiouridine (sU) or sU modified with azidophenylacyl bromide (APAB), either two nucleotides upstream or eight nucleotides downstream from the nucleotide sequence ACC, the codon for tRNA. The sequences of the mRNA analogues were described earlier (Stade, K., Rinke-Appel, J., and Brimacombe, R.(1989) Nucleic Acids Res. 17, 9889-9908; Rinke-Appel, J., Stade, K., and Brimacombe, R.(1991) EMBO J. 10, 2195-2202). Under equilibrium conditions, both of these mRNA analogues bind and cross-link to 70 S ribosomes without the presence of tRNA; however, there are significant increases both in binding and particularly in cross-linking in the presence of the tRNA. Four regions contain cross-linking sites that increase in the presence of tRNA, C, A, A (and minor sites around these three positions), and C/U. Three other cross-linking sites, U, A, and U, show very little change in extent of cross-linking when tRNA is present. A conformational change in the 30 S subunit allowing additional accessibility to the 16 S rRNA by the mRNA analogues upon tRNA binding best explains the behavior of the tRNA-dependent and tRNA-independent mRNA-16 S rRNA cross-linking sites.


INTRODUCTION

Photoaffinity experiments with suitably derivatized mRNA and tRNA substrates offer an attractive strategy to determine the location and properties of the functional sites on the Escherichia coli ribosome. Photo and photochemical cross-links have been made to the ribosome from the acceptor end (1, 2) , from positions in the hinge region (3, 4) , and from the anticodon loop of the tRNA (5, 6) . The general location of the mRNA track has also been elucidated by photoaffinity experiments (7, 8) . These experiments are consistent with an mRNA track extending around the middle part of the 30 S subunit, with the sites for the codon-anticodon interactions determined by the 30 S subunit and with the aminoacyl ends of the tRNAs extending toward the peptidyltransferase center of the 50 S subunit (see Refs. 2 and 9-11).

Experiments from the groups of Brimacombe and Bogdanov (12, 13, 14, 15, 16) have been done with synthetic RNA molecules containing photoreactive nucleoside sU() at different locations with respect to the P site to probe the mRNA binding track. Cross-links to four sites in the 16 S rRNA have been reported: A, C (and residues G and A in its close vicinity), U, and the 3`-terminal region. Several specific correspondences between the location of the photoreactive moiety in the mRNA analogue and the position of cross-linking in the 16 S rRNA have been drawn. If the numbering of positions in the mRNA analogue are made from ``+1'' being the first nucleotide of the triplet used in the P-site, these are +6 in the mRNA to U in the 16 S rRNA, +7 in the mRNA to C in the 16 S rRNA, and +11 in the mRNA to A in the 16 S rRNA (15) . In subsequent reports using additional mRNA analogues, cross-links were detected from +4 in the mRNA to nucleotides in the interval 1402-1415 in the 16 S rRNA, from +6 to +10 in the mRNA to C in the 16 S rRNA, and from +10 to +13 in the mRNA to A in the 16 S rRNA (14) . These cross-links were strongly tRNA-dependent; in all experiments, complexes of 70 S ribosomes, mRNA, and tRNA were isolated by sedimentation or gel filtration to ensure that the most stable complexes were examined.

We reported experiments that used RNA analogues 51 nt in length with clusters of sU in different areas from -20 to +26 from the first nucleotide of the codon (17) . These experiments were done by forming complexes between 70 S ribosomes and mRNA analogues without or with cognate tRNA and irradiating these complexes without further purification. Ten cross-links between the mRNA analogues and 16 S rRNA were detected by primer extension assay. With the mRNA analogues that were used, only two of the sites (C and A) showed strong tRNA dependence and the site A showed a weaker tRNA dependence on cross-linking for some of the mRNA analogues. The behavior of this set of mRNA analogues led us to question why there was not tRNA dependence for a larger number of the rRNA sites.

In the present experiments, the pattern of cross-linking made by mRNA analogues with single sU residues and the response of the sites with respect to tRNA binding has been investigated. mRNA analogues with sU either at position -2 or +11 (12, 13) were used because these were shown to exhibit strong tRNA dependence in binding and cross-linking. Under equilibrium binding conditions, the pattern of cross-linking can be separated into tRNA-dependent and tRNA-independent classes. A proposal for a conformation change in the 30 S subunit upon tRNA binding is made to explain the behavior of the mRNA analogues with the tRNA-dependent 16 S rRNA cross-linking sites.


MATERIALS AND METHODS

Preparation of DNA Template and RNA Synthesis

dsDNA for in vitro transcription was prepared with Taq DNA polymerase. Single-stranded DNA template containing the T7 promoter sequence and the sequence for the mRNA analogue were synthesized, and only left (5`) primers were used since both molecules contain redundant sequences at their 3` ends. dsDNA was purified on nondenaturing 8% polyacrylamide gels. In vitro transcription of RNA was done with RiboMax protocols described by Promega and included 1 mM GMP for partial substitution of GMP for GTP at the 5` end. In vitro transcription of RNA containing sU was done with 0.25 mM sUTP (18) and 3 mM concentration each of GTP, ATP, and CTP. P-Labeled RNA was made by including 20 µCi of [-P]GTP (3000 Ci/mmol, Amersham) in a reaction that had 0.5 mM cold GTP. Usual yields were 40-60 µg of RNA/100 µl transcription. Transcription mixtures were purified on Qiagen tips, and RNA was checked on 8% polyacrylamide, 8 M urea gels. When unlabeled RNA was transcribed, 50 pmol was used in exchange reaction with [-P]ATP (19) .

RNAs containing sU were used for APAB reaction as described (12) . APAB-substituted mRNAs were checked on 8% polyacrylamide, 8 M urea gels containing 0.003% APM (20) in which sU-containing RNA is retarded, but sU-containing RNA substituted with APAB migrates at the same position as RNA with uridine.

Cross-linking and Analysis

Complexes between activated 70 S ribosomes and mRNA analogues were made in 10 mM Tris HCl, pH 7.4, 50 mM KCl, and 10 mM MgCl(21) . 10 pmol of 70 S ribosomes in 100 µl were incubated for 30 min at 37 °C with a 5-fold molar excess of mRNA analogue without or with a 5-fold molar excess of tRNA. mRNA binding was determined by nitrocellulose filter binding as described (17) . The extent of tRNA binding was determined by footprinting reactions (22, 23) comparing the degree of protection conferred by tRNA to that conferred by a known amount of binding of tRNA (directed by poly(U)). Cross-linking was carried out for 10 min at 4 °C in an irradiator producing 320-365 nm light (24) . Irradiated samples were digested with proteinase K, phenol-extracted, ethanol-precipitated, and redissolved in water. Samples were denatured with formamide before electrophoresis on agarose gels. Primer extension analysis of the mRNA-rRNA cross-links using reverse transcriptase and 10 DNA oligonucleotide primers was done as described previously (17, 25) . Reverse transcripts were analyzed on 8% polyacrylamide, 8 M urea gels.

Determination of Cross-linking in the 16 S rRNA 3`-Terminal Region

To determine the extent of cross-linking to P-labeled mRNA at the 3` end of 16 S RNA, 16 S-sized RNA was purified on 1% agarose gels before use for RNase H reactions. The oligomer UACTCCUUUGG, complementary to 1426-1437, consisting of 2`-methyl ribonucleosides and deoxynucleotides (underlined) was used to cleave after position 1435 (26) . Reactions contained 40 mM Tris HCl, pH 7.4, 4 mM MgCl, 1 mM dithiothreitol, 0.003% bovine serum albumin, 4% glycerol, 1 µg of 16 S rRNA, and 50 pmol of oligomer in 6 µl. Reactions were done by incubating the mixture for 2 min at 55 °C, slowly cooling to 37 °C, and then adding 2.5 units of RNase H (Fermentas) and incubating for 10 min at 37 °C. After phenol extraction and precipitation, RNA was electrophoresed on composite 4%-12% polyacrylamide, 8 M urea gels. For sequencing, the 3` ends of cross-linked and control 16 S rRNA were labeled with [5`-P]pCp and T RNA ligase (27) according to Krzyzosiak et al.(28) . RNase H was used to remove the label from the mRNA with additional DNA oligonucleotides complementary to the mRNA. Partial alkaline hydrolysis was done in 50 mM NaCO, pH 9.0, 1 mM EDTA in the presence of 0.25 µg/µl tRNA and 3`-labeled 16 S RNA fragment (0.1 µg) in 6 µl for 10 min at 90 °C, then 4.3 mg of urea were added, and samples were electrophoresed on 8% polyacrylamide, 8 M urea gels. For sequencing, samples were digested with RNase T and RNase U(29) .


RESULTS

mRNA Binding and Cross-linking

The sequences of the two mRNA analogues used in this study are shown in . The stoichiometries of binding to ribosomes under the conditions used for cross-linking were determined by nitrocellulose filter binding and are summarized in . In all cases, more than one mRNA binds per ribosome at saturation. Binding isotherms were examined to estimate dissociation constants assuming independent sites. For the mRNA analogue with sU at position -2 with respect to the sequence ACC (designated sU(-2)), K values of 86 nM (for both sites) without tRNA and 19 nM and 77 nM with tRNA were determined. For the mRNA analogue with sU at position +11 (designated sU(+11)), K values of 104 nM and 178 nM without and 28 nM and 93 nM with tRNA were determined. To test if the presence of the two types of mRNA binding sites reflected ribosome heterogeneity, tRNA binding was measured at an mRNA analogue concentration that resulted in an average of 1 mRNA analogue/ribosome. tRNA binding was found to be at least 0.85 tRNA/ribosome, all confined to the P-site (results not shown). Thus, all ribosomes contain a strong binding site for these mRNA analogues.

Complexes of ribosomes and sU-containing mRNA or sU-containing mRNA derivatized with APAB were irradiated with 320-365 nm wavelength light. Complexes with mRNA analogues containing uridine were irradiated as control samples. Electrophoresis of purified RNA from these samples revealed that mRNA becomes extensively cross-linked to 16 S rRNA with smaller cross-linking to 23 S rRNA ( Fig. 1and I). The presence of tRNA increases cross-linking from 3- to nearly 5-fold in 16 S rRNA for both the(-2) and (+11) mRNA analogues. Cross-linking to 23 S rRNA is independent of the presence of tRNA. Binding and cross-linking of sU-containing mRNA analogues can be competed by uridine-containing mRNA analogues (results not shown).


Figure 1: Agarose gel electrophoresis of RNA after cross-linking reactions between P-labeled mRNA and ribosomes. Experiments are shown with mRNA analogues with uridine or sU at positions -2 or +11 counting from the first position of the sequence ACC (the codon for tRNA). The mRNA analogues contain uridine (U) for control, sU, or sU modified with azidophenylacyl bromide (APAB). Complexes contained tRNA as indicated. The positions of 23 S rRNA, 16 S rRNA, and mRNA are indicated.



Reverse Transcription Analysis

Reverse transcription experiments were done to determine the location of the mRNA cross-links in the 16 S rRNA. The mRNA analogue sU(+11) cross-links in the interval 1389 to 1393 to C (the predominant site) and to C (Fig. 2). The frequencies of all of these increase significantly when tRNA is present as measured as the ratio of the band intensity in the cross-linked sample over that in the control sample (). When sU(+11)-APAB is used in the cross-linking reaction, C and C are still present as prominent cross-linking sites and there is a new site at A. The presence of tRNA slightly decreases the extent of cross-linking by sU(+11)-APAB, except at A where there is a large increase. For mRNA analogue sU(-2), one cross-linking site at U is seen in this region, and this does not change significantly in extent when tRNA is present ( Fig. 2and ). Reactions with the mRNA analogue sU(-2)-APAB show lower amounts of cross-linking at U, presumably because the azidophenylacyl moiety interferes with the mRNA-rRNA interaction at this site, and the appearance of a new cross-link at U. Because the stoichiometry of the mRNA binding exceeded 1 mol/mol 70 S, the experiments were also done with lower mRNA input levels; this reduces the amount of covalent cross-linking but does not change the pattern (results not shown).


Figure 2: Reverse transcription analysis of the pattern of mRNA cross-linking in the 16 S rRNA region 1417-1365. Samples were prepared from cross-linking reactions containing mRNA analogues containing sU, containing sU substituted with APAB (written as APA), or containing uridine and with or without tRNA as indicated. Reverse transcription samples in the left part of the gel (lanes O, A, G, C, and U) were prepared with total un-irradiated RNA from 70 S ribosomes without dideoxynucleotide or with ddTTP, ddCTP, ddGTP, or ddATP. Nucleotide numbering on the right of the gel indicates stopping positions attributed to the presence of mRNA cross-links; the identity of the nucleotide 5` to the stopping site is written.



mRNA analogue sU(+11) cross-links to sites in the 16 S rRNA at A and U with lower intensity sites at A, U, and A (Fig. 3). All of these show increases when tRNA is present. Cross-linking in this region is somewhat greater with mRNA analogue sU(+11)-APAB but, in this case, increases in cross-linking upon tRNA addition are smaller.


Figure 3: Reverse transcription analysis of the pattern of mRNA cross-linking in the 16 S rRNA region 539-515. Sample lanes from sU-containing mRNA analogues, sU-containing mRNA analogue derivatized with APAB (APA), or with uridine-containing mRNA analogue are indicated. The sample O` is a control made with total RNA from irradiated 70 S ribosomes that did not contain mRNA or tRNA. The positions that indicate cross-links at U and A are indicated. For the sample sU(+11) with tRNA, there are also cross-links at A and U.



Three additional regions around A, U, and A contain cross-linking sites (Fig. 4). The sites A and U occur with mRNA analogue sU(-2) and sU(-2)-APAB. All of these show little change in extent when tRNA is present. Finally, a cross-linking site at A occurs in mRNA analogue sU(+11) (Fig. 4C). This site shows the same behavior with respect to tRNA dependence as the sites at C-A and around A. With mRNA analogue sU(+11)-APAB, three additional sites C, U, and G are also seen and these also increase when tRNA is present.


Figure 4: Reverse transcription analysis of the pattern of mRNA cross-linking in three 16 S rRNA regions. A shows the region 860 to 826 containing the cross-linking site at A. B shows the region 748 to 695 which contains the cross-linking site at U. C shows the region 1205 to 1185 which contains the cross-linking site at A. In C, positions C, U, and G are also cross-linking sites for sU(+11) derivatized with APAB. The lanes are indicated as in Fig. 3.



The experiments involving mRNA sU(-2) have also been performed by isolating stable complexes by sedimentation before cross-linking. This decreases the amount of mRNA bound to the 70 S ribosomes particularly in the absence of tRNA as described before (13) . However, it was still possible to detect all cross-links including the tRNA independent sites (U, A, and U) without tRNA although there are much lower levels of cross-linking in those complexes, reflecting the lower levels of bound mRNA in the absence of tRNA (data not shown).

Determination of Cross-linking Sites in the 3`-Terminal Region

The terminal 30 nucleotides of the 16 S rRNA cannot be examined by reverse transcription because of mA at positions 1518 and A. Since Rinke-Appel et al.(13) reported cross-linking to this region with mRNA analogue sU(-2), levels of cross-linking were determined using RNase H cleavage with an oligonucleotide complementary to 1426-1437. If the 16 S rRNA were cross-linked to P-labeled mRNA, a fragment longer than expected would be released and it would be radioactive. This is the case and levels of cross-linking were determined from an autoradiogram of a polyacrylamide gel (results not shown). Cross-linking in the terminal 107 nt occurred only with mRNA analogue sU(-2); in cross-linking reactions without tRNA, the extent was 4%, and, in reactions with tRNA, the extent was 39% ().

RNA sequencing was done to determine the cross-linking site. After cross-linking to nonradioactive sU(-2) and purification of 16 S-sized RNA, RNA was 3`-end-labeled. RNase H cleavage at position 1435 of 16 S-sized RNA from cross-linking reactions releases two prominent products, one of which has the same gel mobility as the 16 S rRNA terminal 107 nt, and the other has a significantly slower mobility (Fig. 5, lane 5). When an oligonucleotide complementary to the mRNA was included in the RNase H digestion to reduce the size of the mRNA and remove its 3` end label, the slower product increases in mobility but still is different from the terminal 107 nt of the 16 S rRNA (Fig. 5, lane 6). The control fragment containing the terminal 107 nt and the fragment containing the cross-linked RNA were isolated and subjected to partial hydrolysis and RNase T and RNase U digestion to find the site of the cross-link. In this comparison, the cross-linked sample and control sample should show the same pattern up to the last nucleotide before the cross-linking site. The patterns are very similar to position C and then the pattern in the cross-linked sample contains a gap (Fig. 6). The intensity of the partial hydrolysis product at C is significantly less than expected indicating a cross-link at C and the band expected at U is completely absent. Given the sequence of the residual mRNA fragment still attached to the terminal 107 nt of the 16 S rRNA, the patterns of partial RNase T and RNase U digestion are consistent with the assignment. Densitometry was done to estimate the amount of cross-linking at each of these sites (). The location of these and the other cross-linking sites are indicated in the 16 S rRNA secondary structure in Fig. 7.


Figure 5: Analysis of cross-linking to the 3`-terminal region of 16 S rRNA using RNase H digestion. Control 16 S rRNA or 16 S rRNA from ribosomes cross-linked with mRNA analogue sU(-2) were purified, 3`-end-labeled, and then subjected to RNase H digestion using oligonucleotides complementary to 16 S rRNA (positions 1426-1437) and mRNA (positions) as indicated. The gel used was a denaturing gel of two parts: 4% polyacrylamide on top and 12% polyacrylamide on bottom; sections showing the 16 S rRNA and relevant fragments are shown. RNA from the bands containing the 107-nt fragment from control 16 S rRNA and containing the terminal 107-nt fragment cross-linked to the residual mRNA was isolated for sequencing analysis.




Figure 6: Partial hydrolysis reaction on the 3`-terminal region of 16 S rRNA to determine the point of mRNA cross-linking. The control RNA fragment and RNA fragment containing the mRNA cross-link were subjected to partial hydrolysis (H), partial digestion with RNase T (T), or RNase U (U) as indicated. The band that corresponds to the fragment whose 5`-nucleotide is C is indicated.




Figure 7: Location of mRNA cross-linking sites in the 16 S rRNA secondary structure (49). The mRNA cross-linking sites that change in frequency by more than 50% in the presence of tRNA are shown by solid dots, and those that are independent of tRNA are indicated by open dots. All of the cross-links are made by sU at (-2) or (+11) in the mRNA analogues except those designated with an asterisk which only appeared if the sU was additionally derivatized with APAB.




DISCUSSION

Several conclusions made by Stade et al.(12) and Rinke-Appel et al.(13) for cross-linking by these two mRNA analogues to E. coli rRNA have been confirmed, including the strong tRNA dependence for cross-linking, cross-linking to C and A by the mRNA analogue sU(+11), and cross-linking to the 3`-terminal region by mRNA analogue sU(-2). There are additional cross-linking sites in the neighborhoods of these prominent cross-links detected by reverse transcription (, Fig. 7). Subsequently, a cross-link at U was reported from position +6 in another mRNA analogue (14) . We do not detect U but instead detect A (this report and Ref. 17) which is very close to U in the secondary structure; these probably represent the same mRNA-16 S rRNA interaction region.

Three cross-linking sites in the 16 S rRNA at U, U, and A are also detected. All of these sites are made by mRNA analogue sU(-2) and its APAB derivative and are nearly tRNA independent. These sites as well as the tRNA-dependent cross-linking sites have also been detected using other mRNA analogues (17) which were both longer and lower in purine base composition than the molecules sU(-2) and sU(+11). An apparent positional ambiguity of the mRNA analogues in the mRNA track was determined in our previous experiments due to the possibility of conformational flexibility in the 30 S subunit (17) . The same phenomenon may occur with these two mRNA analogues; thus, although the mRNA analogue sU(+11) makes cross-links with A, A, and C, this does not necessarily mean these sites are direct neighbors of one another.

An important consideration involves the strategy of performing these experiments. The Brimacombe and Brimacombe/Bogdanov groups used sedimentation or gel filtration to first purify mRNAribosome complexes before cross-linking, and this decreases the amount of bound mRNA particularly in those complexes formed without tRNA. In our case, ribosomes in equilibrium with unbound mRNA and tRNA typically were irradiated, allowing a larger extent of mRNA binding and cross-linking both with and without tRNA. This has allowed the recognition of both tRNA-dependent and tRNA-independent cross-linking sites. The dependence of mRNA binding on tRNA is consistent with the behavior of poly(U) and tRNA under subsaturation conditions observed by Katunin et al.(21) . Weller and Hill (30) have shown recently that competition between oligo(U) and a DNA oligonucleotide complementary to 16 S rRNA interval 1396-1403 occurs only if tRNA is present, again indicating a cooperativity between mRNA and tRNA binding.

The location of the mRNA cross-linking sites in the 30 S subunit can be estimated from available structural information. The site A has been associated with protein S4 by a number of experiments (31, 32, 33) , and S4, in turn, has been located in the indentation between the head and body of the 30 S subunit on the side away from the platform region (34) . Sites close to A have been associated with protein S5 (see Ref. 35). S5 has been located in the middle of the subunit on the cytoplasmic side, close to S4 (34) . The sites at U to C are in the vicinity of the codon-anticodon interaction in the cleft region evidenced by the anticodon-rRNA cross-link at C in the 16 S rRNA (5) and the determination of this site by electron microscopy (36) . Likewise, the site at C/U is in the vicinity of the codon/anticodon interaction because of cross-links between this region and proteins S7, S18, and S21 (32) and the locations of these proteins (34) . Thus, the four tRNA-dependent sites break down into two groups: the 532 and 1196 regions in the S4 subdomain and the 1395 and 1533 regions close to the P-site codon/anticodon interaction. There has been some differing information about the closeness of these two regions. On the one hand, a large distance between protein S4 and S7 has been determined by neutron scattering (34) and electron microscopy (37) . On the other hand, there is evidence intimately linking the 530 region with the decoding process including evidence that tRNA in both the A-site and P-site produces footprints in the interval 529-532 (23) , that nucleotide changes in the 523 to 530 region strongly affect translational proofreading and ribosome function (38-42), and that the 530 region is involved in EF-Tu function (43, 44) . In addition, Alexander et al.(45) have recently shown that protein S7 and the 3`-terminal region of the 16 S rRNA can be labeled by a photoaffinity probe annealed to the 16 S rRNA in the interval 518-526, indicating a distance of no greater than 24 Å between C and the 16 S terminal region and protein S7.

The tRNA independent mRNA cross-linking sites can also be placed in approximate locations in the 30 S subunit. The cross-linking site U is in the region associated with protein S7 (32, 46) the location of which has been determined on the side of the head of the subunit facing the cleft region. The cross-linking sites U and A are in the rRNA regions associated with S6, S18, S15, and S8 (32) , and these have been located in the side of the body next to the decoding region (34) . There are large distances between S7 and the other group of proteins suggesting that this part of the mRNA track is quite wide.

The properties of the tRNA-independent and tRNA-dependent cross-linking indicate a change in the 30 S mRNA analogue interaction upon tRNA binding. First, under the experimental conditions, mRNA binds with reasonable stoichiometry to 70 S ribosomes whether tRNA is present or not. However, there is a large increase in the amount of covalent cross-linking to the 16 S rRNA upon inclusion of tRNA, much larger than the binding stoichiometry differences. As an internal control, there is no significant difference in the amount of cross-linking to 23 S rRNA. Second, the mRNA bound to the 70 S ribosomes is bound in approximately the correct location without tRNA, and cross-links occur to all of the rRNA sites (see ). The Shine-Dalgarno sequence homologies in the mRNA analogues probably are responsible for the correct alignment of the mRNA analogues in the ribosome.

There are several ways to account for the large increase in cross-linking efficiency upon tRNA binding. One possibility would be an increase in mRNA flexibility upon tRNA binding since sU cross-linking efficiency is greater at interactions that have structural mobility (47) , but this would be contrary to the increase in mRNA binding strength and the fact that the mRNA derivatized with APA behaves in a similar manner. Another possibility is that tRNA binding subtly alters the orientation of the mRNA analogue on the subunit. A specific change in the arrangement of sU at -2 with respect to the U/C site may occur because tRNA binding, together with the Shine-Dalgarno interaction, would constrain the mRNA. However, a constraint imposed by tRNA binding is unlikely to account for the behavior of sU(+11) because of the distance between sU at position +11 and the ACC codon. The last possibility is that there is a conformational change in the 30 S subunit upon tRNA binding that results in greater exposure of the reactive 16 S rRNA sites. This would account for the increase in cross-linking of sU at position +11 as well as at position -2 and would also be consistent with the increase in mRNA binding strength, if there is an associated increase in favorable mRNA-30 S subunit interactions. Since the tRNA-dependent mRNA cross-links are located at the interface between the body and head of the 30 S subunit, some movement of the head of the 30 S subunit may be involved in this change to alter the accessibility of these rRNA sites. Huttenhofer and Noller (48) recently determined the protection of mRNA analogues on the 30 S subunit from hydroxyl radical cleavage and found that the protection increased from about 40 nt to 54 nt when tRNA was present. Their interpretation was that the codon-anticodon interaction influenced the mRNA pathway, particularly past position +6 from the codon. Their observation is also consistent with a tRNA-dependent strengthening and alteration in the mRNA-30 S subunit interaction indicated by the present cross-linking results.

  
Table: Sequences of the mRNA analogues sU(-2) and sU(+11)

Molecules sU(-2) and sU(+11) are the same sequences as the molecules ``mRNA No. 1'' from Stade et al. (12) and ``I.'' from Rinke-Appel et al. (13). Numbering of the positions in the mRNA analogues is from the first nucleotide of codon for tRNA.


  
Table: Stoichiometry of binding mRNA sU(-2) and sU(+11) to 70 S ribosomes without and with cognate tRNA

Stoichiometry of binding (mol mRNA analogue/mol 70 S ribosome) were determined at 5 molar excess of mRNA analogue over 70 S ribosome. When present, tRNA was at 5 molar excess over 70 S ribosomes.


  
Table: Extent of cross-linking mRNA analogues sU(-2) and sU(+11) to 70 S ribosomes without and with tRNA

Extent of cross-linking to 16 S rRNA and 23 S rRNA is indicated as percent mol mRNA analogue/mol rRNA species.


  
Table: Summary of the 16 S rRNA cross-linking sites and relative extent of cross-linking

Entries indicate the normalized relative intensity of bands for cross-linked versus control samples. The two values for each are cross-linking without and with tRNA. For all cross-linking sites, it was assumed that reverse transcriptase stops completely at the 3`-nucleotide to the cross-link. Positions that had an increase in relative band intensity values of at least 1.5 either without or with tRNA are listed. Empty areas indicate no cross-links under any condition. ND, is not determined. The most frequent cross-linking site in each group is underlined.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM43237 and North Carolina Biotechnology Center Grant 9107-PIG-7007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institute of Biochemistry, Lithuanian Academy of Sciences, Vilnius, Lithuania.

To whom correspondence and reprint requests should be addressed. Tel.: 919-515-5703; Fax: 919-515-2047.

The abbreviations used are: sU, 4-thiouridine; APAB (or APA), azidophenylacyl bromide; APM, [(N-acryloylamino)phenyl]mercuric chloride; dsDNA, double-stranded DNA; nt, nucleotide(s); TBE, 89 mM Tris, 89 mM boric acid, 10 mM EDTA.


ACKNOWLEDGEMENTS

We thank Melanie Blackard for providing the mixed oligomer 16 S-OneB and conditions for its use.


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