From the
Memorial Sloan Kettering Cancer Center,
Cellular Biochemistry and Biophysics Program, New York, New York 10021, the
Max-Planck-Institut für Molekulare Genetik,
AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany, and the
||Department of Biological Sciences, University of
Maryland, Baltimore County, Baltimore, Maryland 21250
Received for publication, March 14, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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R-protein L4 from Escherichia coli specifically regulates the S10 operon, which codes for 11 r-proteins including L4 itself (3, 4). L4 is unique among the regulatory ribosomal proteins because it regulates not only translation but also transcription of the S10 operon mRNA. The latter form of regulation is accomplished by L4 stimulation of transcription termination at a terminator (attenuator) structure in the mRNA upstream of the initiation codon of the first gene of the operon (5). Determinants necessary for L4-mediated autogenous control are contained within a 172-nucleotide 5'-untranslated region of the S10 operon mRNA. This region folds into six stem-loop structures termed helices HA, HB, HC, HD, HE, and HG (Fig. 1A) (6, 7).
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Binding of a regulatory r-protein to specific targets in both rRNA and mRNA is fundamental to the autogenous control model (13). The minimal rRNA target of L4, identified by serial selection of random E. coli rRNA fragments binding to L4 (14), is a four-helix junction composed of nucleotides 295343 in Domain I of the 23 S rRNA. Binding of protein L4 to this RNA sequence generates a characteristic iodine cleavage pattern in the U321-loop of 23 S rRNA (15). Interestingly, the same region of 23 S rRNA competes with the paused transcription complex for L4, eliminating the ability of the protein to stimulate transcription termination (16). These results show that binding of 23 S rRNA to L4 inactivates the regulatory capacity of the protein and suggest that the 23 S rRNA and mRNA binding sites for L4 might share certain critical features. However, mutations in protein L4 can distinguish its ability to regulate the expression of the S10 operon from its ability to be incorporated into the ribosome (17).
Here we demonstrate direct binding of r-protein L4 to the S10 mRNA leader. Furthermore, we analyze the requirements in the RNA binding site for L4 binding. Footprinting results indicate structural changes of the leader RNA upon binding, and reveal structural similarities between the rRNA and mRNA L4 binding sites. A structure model for the L4-mRNA interaction is proposed.
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EXPERIMENTAL PROCEDURES |
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In Vivo Regulatory StudiesThe regulatory capacity of the
T. aquaticus L4 proteins was determined as described
(7). A C-terminal 6-histidine
tag (no thrombin cleavage site) was introduced into both L4TH (wt) and L4THD7
genes, and the genes were placed under control of an arabinose-inducible
promoter in a pBAD18 derivative. The plasmids were introduced into E.
coli K12 strain LL308 containing target plasmid
pACYC-S10'/'lacZ or
pACYC-HD-S10'/'lacZ. Plasmid
pACYC-S10'/'lacZ contains the entire E.
coli S10 leader and proximal 54 codons of the S10 structural
gene, fused in-frame with a lacZ gene (lacking the proximal 8
codons), and under control of the
isopropyl-1-thio-
-D-galactopyranoside-inducible trc
promoter. This plasmid contains the regulatory signals necessary for both
transcription and translation control of
-galactosidase synthesis by
r-protein L4 (19). Plasmid
pACYC-
HD-S10'/'lacZ is a derivative of
pACYC-S10'/'lacZ that has a deletion of the S10
leader hairpin HD eliminating L4-mediated transcription control
(11).
S10 Leader mRNA ConstructsPlasmid pT724 (which encodes the entire S10 operon leader and proximal one and a half structural genes (6)) was used as PCR template to amplify various parts of the S10 leader mRNA. The PCR products were cloned into the BamHI/EcoRI site of pSP65 (Promega). The 5'(+) primer introduced a T7 promotor, and the 3'() primer introduced a restriction site for linearization of the plasmid for run-off T7 RNA polymerase transcription (20). We used constructs with transcription starts at four sites of the leader: (i) at HA with G1 as the first nucleotide, (ii) at HE with the start at GGG-C86, (iii) at HD with the start at GG-A62, and (iv) at HD with the start at GG-A64 (Fig. 1, green arrows). The two nucleotide differences in the latter two constructs had no effect on the experimental results reported. The 3' termini of the RNA transcripts were generated by digestion of the templates with restriction enzymes SspI (after helix HC at U66), SnaBI (after HD at C86), BsaI (in the lower HE at C100), HpaI (C125 after the upper HE at C125), DraI (after HE at U147), or SmaI (after HG at C197) (Fig. 1A, red brackets). We also made a construct encoding a transcript with a hammerhead generating the C125 end.
Nitrocellulose Filter Binding AssaysNitrocellulose
filtration assays, described in Ref.
20, were done in 300-µl
reactions containing 20 mM Hepes-KOH (pH 7.5), 4 mM
MgCl2, 4 mM -mercaptoethanol, 200400
mM NH4Cl, RNase-free BSA and tRNA bulk (as indicated).
Binding data were fitted to single site hyperbolic curves
(Fig. 1, upper panel).
In competition assays, the L4 concentration was 0.67 µM and
32P-labeled RNA AG (5001500 dpm/pmol) was 0.33
µM. Under these conditions about 22 pmol of complex was
collected on the nitrocellulose filter. Competitor (i.e. non-labeled)
RNA was added to labeled RNA AG before the refolding step. Competition
data were fitted with the program Sigma Plot according to Equation 5 of Lin
and Riggs (21) solved for the
fraction of labeled RNA bound (Fig.
1, middle and lower panel).
Gel Shift AssaysA 15-µl sample of an RNA-protein complex containing 75 pmol of [32P]RNA and the indicated amount of protein L4 was prepared by incubating for 15 min at 37 °C (20 mM Hepes-KOH (pH 7.4), 100 mM NH4Cl,4mM MgCl2, 10% glycerol) and then chilling on ice. The sample was loaded onto a buffered (20 mM Hepes-KOH (pH 7.4), 100 mM NH4Cl, 4 mM MgCl2) 5% acrylamide/bisacrylamide (29/1) gel (0.75 x 130 x 170 mm). The gel was run at 110 V for2hat4 °Candthe electrode buffer was recirculated between the electrode chambers with a pump. The gels were dried and exposed on a phosphoimaging plate (Fuji) for quantification.
Iodine FootprintingThe experiments were done as described
previously for the complex of ribosomal RNA with protein L4
(15). Phosphorothioated RNA
was produced by including Sp-[-S]NTPs (PerkinElmer
Life Sciences and Glen Research) in the T7 RNA polymerase transcription assay.
The L4 concentration was 0.67 µM and that of
32P-labeled phosphorothioated RNA was 0.33 µM or vice
versa (the yield of complex was identical in both experiments). As seen in
some experiments, the mRNA fragments tend to break at C38-A39 and C63-A64 even
in the absence of iodine, and at G80-A81 in the presence of L4THD7. However,
in general control experiments carried out without addition of iodine did not
result in cleavages.
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RESULTS |
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Because many highly positively charged ribosomal proteins, such as L4, bind with considerable strength to nonspecific RNAs (e.g. Refs. 25 and 26), we tested the specificity of L4 binding to the S10 leader by competition experiments (Fig. 2, middle panel). As expected, non-radioactive S10 leader RNA (AG) competes with binding of radioactive leader RNA. In contrast, addition of a 218-nt mRNA molecule that contains the interaction site for r-protein S15 in the translational regulation of the rpsO operon (27) did not affect complex formation between the cognate S10 leader mRNA and L4. Thus, the affinity of L4 for the S10 leader mRNA is significantly higher than its affinity for a noncognate r-operon mRNA, indicating that the L4-S10 leader interaction is specific. On the other hand, the rRNA binding site of ribosomal protein L4 (RNA-DI) strongly competes with binding to the S10 leader mRNA (Fig. 2, middle). Not assuming allosteric effects, this observation is consistent with the hypothesis that the L4 binding sites on 23 S rRNA Domain I and the S10 leader share significant structural features.
To define the binding site more precisely, we tested various S10 leader mRNA subdomains in the competition experiments (Fig. 2, bottom panel). Most intriguingly, the mRNA fragments that do not contain helix HD, such as AC (66 nt), E (65 nt), and EG (116 nt), do not inhibit complex formation, whereas the isolated helix HD (D, 27 nt) competes significantly. All other constructs containing HD (viz. AD (86 nt), DE (88 nt), and DG (139 nt)) compete to the same extent as the full-length AG (198 nt). Consistent with data indicating the importance of helix HD in L4-dependent transcription regulation (11, 28), these experiments suggest that helix HD contributes the most to the specificity of L4 binding.
T. aquaticus Protein L4 Actively Regulates E. coli S10 Operon ExpressionProtein L4 from E. coli is not well suited for in vitro binding experiments, because (i) it has to be purified under denaturating conditions either from ribosomes or from inclusion bodies, (ii) it is relatively unstable, (iii) the solubility even in high salt buffers is very low (<50 µM), and (iv) it aggregates even at low concentrations. For example, in gel-shift experiments RNA-L4EC complexes form aggregates that remain for the most part in the gel pocket (not shown).
To circumvent these problems we combined two strategies to develop a better behaved protein. First, because L4 proteins from many other eubacteria regulate the E. coli S10 operon (7, 19), we switched to L4 from T. aquaticus (L4TH), which is 34% identical and 50% similar to L4 from E. coli (Fig. 1B). Proteins from such thermophilic bacterial species have proven to be beneficial for other biochemical and biophysical experiments. Second, we deleted 46 amino acids that, in the ribosome, form an extension protruding into the core from a globular domain located on the surface of the ribosome (29, 30) (cf. Figs. 1B and 4C). In free L4 protein (from Thermotoga maritima) the corresponding amino acids are not organized into a particular structure (31), and hence might contribute to the aggregation problem. Indeed, the deletion of this region in E. coli L4 generates a protein that retains its capacity for regulating the S10 operon, but is more soluble than the full-length protein.2 Therefore, we constructed similar deletion derivatives of the T. aquaticus L4 and screened them for solubility and thermostability. A deletion between Gly45 and Pro92 of L4TH (corresponding to Gly42Pro89 in L4EC) rendered the protein (called L4THD7) very soluble and stable.
Before using L4THD7 for S10 mRNA binding studies we confirmed that the protein is active in inducing autogenous control in E. coli. First, E. coli cells carrying plasmids with either L4TH or L4THD7 under control of an arabinose-inducible promoter could not form colonies on plates containing arabinose, presumably because the induced T. aquaticus L4 proteins reduce expression of the 11 r-protein genes in the E. coli S10 operon, thereby blocking synthesis of ribosomes. Second, we analyzed the ability of the T. aquaticus L4 proteins to inhibit expression of S10'/'lacZ reporter genes. Induced synthesis of the full-length L4TH protein or the deletion protein (L4THD7) reduced about 3-fold the expression of an S10'/'lacZ gene downstream of the wild type S10 leader (Table I). This result demonstrates a substantial regulatory effect of the T. aquaticus L4 proteins, although the regulation is less than that of the native L4 protein. Moreover, a target plasmid containing a deletion of leader hairpin HD, which is required for E. coli L4-mediated transcription control, was much less responsive (Table I), suggesting that the L4TH proteins regulate transcription, and, like E. coli L4, require the HD hairpin. Taken together, these results show that L4THD7 is a valuable analogue of the E. coli L4 with respect to repression of the E. coli S10 operon and has the desired solubility characteristics. Highly purified protein L4THD7 was therefore used for further in vitro studies.
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RNA Determinants for L4 BindingTo determine a minimal binding site in the S10 leader mRNA, RNA leader fragments were assayed for binding to L4THD7 in gel shift assays (Fig. 3). S10 leader mRNA fragment D (GG6286) does not bind to protein L4 in the gel shift experiment, and there is almost no binding to the D-loE fragment (GG64100). The presence of helix E appears to be more important in the band shift assays than apparent from the nitrocellulose filtration experiments (cf. Fig. 2, bottom), because a little binding activity is seen with the complete helix E stem-loop structure (E, GGG86147). Nevertheless, the minimal mRNA that shows high affinity binding is D-upE (64 nt), comprising nucleotides GG64125 (Fig. 3, top). The binding of the D-upE part to L4THD7 determined by nitrocellulose filter binding is shown in Fig. 1 for comparison (upper panel, black squares). Binding is about 2-fold weaker than binding of L4EC to the full-length mRNA fragment.
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Together with HD, the upper helix HE (nt 100125) is strictly
required for high affinity L4 interaction. The D-upE mRNA fragment,
which shortcuts helix HE by connecting C100 and G132 with a UUGC-tetraloop
(Fig. 1A), does not
bind (cf. DE and D-
upE in
Fig. 3, middle). The
primary sequence requirements of the upper HE are not strict, because
replacement of the upHE-loop with an U1A-loop
(Fig. 1A) retains
significant affinity for L4 (D-U1AupE in
Fig. 3, middle).
The gel shift experiments with the L4THD7 are in good agreement with the nitrocellulose filtrations where full-length L4EC has been used, although the apparent dissociation constants are higher. However, gel shift assays are known to generate apparent KD values up to 1 order of magnitude higher than filter binding assays (32). Similar to the nitrocellulose competition experiments presented above, AD and DG bind to L4THD7 in the gel shift system (not shown). Furthermore, binding is stronger with the cognate ribosomal binding site (RNA-DI) and no binding can be detected with the dispensable AC part of the S10 leader mRNA (Fig. 3, bottom). In summary, a minimal 64-nt fragment comprising HD, the ascending side of HE, and the upper HE helix is sufficient for high affinity binding of protein L4.
L4-S10 Leader mRNA Contacts Monitored by Iodine
FootprintingIodine cleavage of phosphorothioated RNA is a useful
tool for examining in detail RNA-protein interactions
(15,
33,
34). To execute such
experiments one -phosphorothioate nucleotide (A, C, G, or U) per
5'-32P-labeled transcript molecule, on average, was
introduced to RNA via T7 in vitro transcription. The phosphorothioate
S10 leader transcripts were used for two types of experiments. In an
interference experiment, RNA-protein complexes were formed with
phosphorothioate-containing RNA and isolated by binding to nitrocellulose
filters. The RNA was then extracted, cleaved with iodine, and subjected to gel
electrophoresis. A band that is weakened or missing on the gel indicates that
the presence of a phosphorothioate moiety at the corresponding position
impairs or prevents the formation of the RNA-protein complex. In a
protection experiment, the RNA is cleaved in the complex preceding
its isolation. A weakened or absent band in this experiment indicates that
iodine does not have free access to the corresponding phosphorothioate in the
backbone of the RNA, i.e. the position is protected from iodine
cleavage. In this way protein interactions with the non-bridging Rp-oxygens of
the RNA phosphate backbone can be mapped with atomic resolution.
Full-length S10 leader AG and the two S10 leader fragments AD and DG were probed in complex with protein L4EC. Additionally, protein L4THD7 has been footprinted in complex with the minimal D-upE mRNA fragment, resulting in a pattern similar to that obtained with protein L4EC on the larger fragments.
Fig. 4A shows a section of the iodine cleavage pattern of the DG region that contains all determinants necessary for transcription and translation control by L4. Taken together, all signals are located in helices HD and HE clearly monitoring the L4 binding site. Protein L4EC shows strong interference with 5'-phosphorothiolated U75, C76, C86, and to C94 (Fig. 4B, stars), and weaker interference at the positions C72, A73, G78, and A99. These same regions are also protected. Non-bridging Rp-oxygens 5' to the following nucleotides are protected against iodine cleavage in the complex with L4EC: U66, G67 (note: not shown in Fig. 4A), U69, and A74 in the 5'-strand of helix HD; in A81/85, C86-U88, A90-G95, and U97-A99 starting from HD extending to the 5' strand of lower helix HE and, finally, C111, C115, U117, U124, and G132 (not shown) in the upper helix HE (Fig. 4B, dots). The signals cover almost every phosphate in the backbone of the 5' part of loHE up to C100. This extensive protection could either be due to direct protein contact in this region or to structural changes induced by L4 binding to the HD region (see "Discussion").
However, the footprint in the loop of helix HD displays very characteristic protein-RNA contacts. Interestingly, the two interference sites 5' and 3' to U75 (Fig. 4B, red stars) are strongly reminiscent of the footprinting pattern of rRNA-DI in complex with L4, where O to S substitutions 5' and 3' to U321 abolish the binding of L4 (14) (Fig. 4B, inset). The three phosphates 5' to A74 and A320 and G78 and A324 are protected in the mRNA and the rRNA, respectively (Fig. 4B, red dots). Although the secondary structures are different, these data raise the intriguing possibility that helix HD of the S10 leader is a close three-dimensional mimic of the rRNA binding site (U321-loop, Fig. 4B).
Mutational Analysis of the HD LoopBecause the footprinting
data suggested direct L4 binding to the loop of hairpin HD, and because the
pattern was strongly reminiscent of the interaction of L4 with its rRNA
target, we analyzed the sequence requirements for L4 interaction with hairpin
HD in more detail. Comparison of the E. coli HD loop structure with
the homologous structure in S10 leader mRNAs from other -proteobacteria
(7), and with the U321 loop in
ribosomal RNA (L4-binding site) of different species reveals the following
(Fig. 5). (i) A74 in the E.
coli mRNA, which corresponds to position 320 in rRNA, is conserved in the
S10 leader of other proteobacteria that bestow L4-mediated autogenous
control; (ii) the rRNA bases 319 and 323 universally form a Watson-Crick base
pair (e.g. GC in E. coli and Haloarcula
marismortui, and CG in T. thermophilus and
Deinococcus radiodurance), whereas the corresponding bases at
positions 73 and 77 in the mRNA occur as noncanonical AG or GU
combinations; and (iii) the nucleotide corresponding to C76 in E.
coli mRNA is an adenine in 23 S rRNA throughout all three kingdoms of
life (A322 in E. coli). Guided by these comparisons
(Fig. 5) and assuming that the
two L4 binding sites form similar structures, we constructed 14 mutant mRNA
minimal versions and tested them for binding to protein L4THD7 in the band
shift system. Selected experiments are shown in
Fig. 5, together with a
quantitative representation of the binding experiments in
Table II.
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The binding data support the idea that L4 recognizes specific primary or secondary structure features in the HD loop region. First, substitutions of the conserved A at position 74 decrease the binding affinity (Table II, mutants M13), particularly if the base is changed to U (Fig. 5). Second, we were interested in nucleotides 73 and 77 in the HD loop, because they are Watson-Crick paired in the 23 S rRNA target. Interestingly, the affinity for L4 for the leader was significantly reduced when bases in those positions were changed to Watson-Crick combinations (Table II, mutants M58), but additional changes such as C76A (M9 which resembles E. coli rRNA) or changes weakening the loop-closing C72G78 base pair (M1014) partially or fully restored binding affinity. The C76A mutation assayed by itself increased binding slightly above wild type (M4).
In summary, base exchanges in the loop of helix HD modulate the affinity of the S10 leader mRNA to L4 as much as 34-fold. Consistent with in vivo data suggesting that A74 is important, and that requirements for the HD sequence are flexible (28), none of the changes completely eliminates L4 binding. The mutational analysis reflects the phylogenetic conservation of the two RNA sites.
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DISCUSSION |
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The current investigation therefore focuses on analyzing L4 interaction with the S10 leader. To circumvent the difficulties discussed above we used competition strategies and developed an L4 protein to overcome aggregation and precipitation problems. The construction of this protein, derived from T. aquaticus L4, took advantage of the superior stability of r-proteins from thermophiles, and the fact that the long loop covering about 50 amino acids in L4 (cf. Fig. 4C) (29, 30) apparently contributes to the undesirable properties of the protein, but is dispensable for regulation (17).2 The resulting protein, L4THD7, with a 46-amino acid deletion in the extended loop, facilitated biochemical experiments. It also regulated the E. coli S10 operon in vivo, apparently by the same mechanism as E. coli L4 (Table I).
The ability of T. aquaticus L4 to regulate the E. coli S10 operon is interesting, because the T. aquaticus S10 operon is fused with the upstream str operon, and hence has no S10 leader (36). One must therefore assume that L4TH does not regulate the expression of the S10 operon in T. aquaticus, at least not by the mechanisms described for E. coli, yet the protein has maintained the determinants for that function. Similar findings have previously been made for L4 proteins from bacterial species such as Pseudomonas aeruginosa and Bacillus stearothermophilus, species that also lack an E. coli-like S10 leader (7, 19). Taken together, these findings suggest that determinants necessary for the interaction of L4 with the S10 operon mRNA of E. coli are conserved in other eubacteria, we presume because these same determinants are involved in ribosome assembly and/or function in the native background. This conclusion is consistent with our hypothesis that the targets of L4 in the S10 mRNA and in 23 S rRNA share structural features.
The S10 Leader mRNA Binding Site of Protein L4 In this study we demonstrated for the first time direct and independent binding of L4 to the S10 leader RNA, using both nitrocellulose filter competition binding assays (Fig. 2) and gel shift experiments (Fig. 3). The minimal binding site for L4 binding consists of a 64-nt fragment containing hairpin HD, the 5' part of the lower HE, and the stem loop of the upper HE (Fig. 3, top). We propose that L4 binding to the leader requires specific interactions with the HD loop and non-sequence specific interaction with parts of HE.
The requirement for HD is supported by the following results: (i) no leader fragment lacking hairpin HD has significant binding or competition activity (Figs. 2 and 3); (ii) HD competes by itself against binding to other, longer leader fragments and thus seems to contribute the most to the specificity of binding (Fig. 2, bottom); and (iii) point mutations in the loop of hairpin HD positively and negatively alter binding affinity (Fig. 5, Table II). Thus, hairpin HD is essential for binding and seems to be the main determinant for specificity. This result is consistent with in vivo genetic experiments showing that L4 transcription control is abolished upon deletion of hairpin HD (Table I and Ref. 11).
Nevertheless, HD does not bind by itself, so other sequences must
contribute to binding. Because both AD and DG bind
(Fig. 2), this extra energy can
apparently be provided by sequences on either side of HD. The relatively weak
effect of substituting the loop of HE by the U1A-loop (D-U1AupE, a change of
11 bases) implies that these sequences can add binding energy but do not
contribute significantly to specificity. Deletion of the upper HE part, on the
other hand, abolishes binding (D-upE,
Fig. 3, middle). This
requirement might be because of indirect effects, e.g. improper
folding of the actual L4 target, as the upper HE is not protected by L4
binding. Consistently, other hairpins with an intact stem can substitute for
the native upper HE hairpin with little or no loss of activity in
vivo (8,
28). Nonspecific interactions
have also been observed to be an essential part of specific binding and
function in other RNA-protein complexes (e.g. Ref.
37).
Structural Mimicry between mRNA and rRNAThe phosphorothioate footprinting results further define the hairpins HD and HE as the binding target of r-protein L4 (Fig. 4). Surprisingly, almost every phosphate in the backbone of the 5' part of loHE up to C100 is protected. We cannot differentiate whether the backbone is directly shielded from iodine cleavage by protein L4 or the footprint indicates changes in the RNA structure. We favor the latter possibility because it is difficult to see how the protein could protect one strand of the lower helix HE for more than one turn. Rather, the results suggest that in complex with L4, even in fully transcribed RNA and in the absence of an RNA polymerase complex, lower helix HE as such does not exist (cf. Fig. 1A versus 4B), and that its structure is influenced by L4 binding. Notably, secondary structure probing results obtained with the free RNA have been ambiguous for this particular region (6).
On the other hand, the footprint in the loop of helix HD is particularly striking because the pattern is similar to the signals that characterize the interaction of L4 with the U321-loop of 23 S rRNA (14). The rRNA binding site of L4 contains a four-helix junction (RNA-DI, Fig. 4B), which, in the ribosome (29, 30, 38), forms two longer bent helixes: helices H18 and H20 stack on each other generating one continuous helix, whereas H19 stacks on the U321-loop to form the other (Fig. 4C). This way three non-consecutive RNA strands (color coded in Fig. 4, B and C) contribute bases to the helix-like arrangement that is recognized by L4: U304G301 (green) and C334G333 from H20 (red) base pair with G313C318 (blue). Stacking on top of this helix stem, the G319C323 pair closes the three-base U321-loop (cyan). Our interference results raise the interesting possibility that helix HD of the S10 leader RNA has a three-dimensional structure similar to the structure of the H19U321-loop part of the four-helix junction: the two interference sites 5' and 3' to U75 resemble the phosphates 5' and 3' to U321 in 23 S rRNA, where O to S substitutions abolish L4 binding, and the preceding three phosphates are protected, as is the phosphate 3' to U75 and U321 in mRNA and rRNA, respectively (Fig. 4, red dots and stars). The hypothesis that hairpin HD is a three-dimensional structure mimic of the rRNA-DI binding site is supported by our observation that binding of mRNA and rRNA are competitive.
The Watson-Crick 319323 pair closing the three-base U321-loop in rRNA (Fig. 4D) is phylogenetically conserved in all three domains of life (Fig. 5), whereas the phylogenetic analysis of the mRNA suggests that the HD loop has five or more nucleotides (7). In an attempt to change the mRNA structure to a structure more like the rRNA, we changed nt A73 and/or G77 to form Watson-Crick base pairs. Most pronounced in the pyrimidine-purine combinations (i.e. M5 and M8), mutations predicted to form 7377 Watson-Crick base pairs in the HD loop negatively affect the interaction with L4 (Fig. 5, Table II, M5M8). In the crystal structures of the 50 S ribosomal subunit, the 3' nucleotide of the Watson-Crick pair (C330 of H. marismortui (38) and G334 of D. radiodurans (30)) is in a syn-conformation causing a turn between this and the preceding base (Fig. 4D). It appears difficult to form a Watson-Crick base pair with one base in the syn-conformation in the context of an A-form helix like HD, because it would similarly require a local reversal of strand direction (39).
Whereas keeping the G73C77 Watson-Crick pair, we tried to provide a context to form a three-base loop with a similar conformation as in the rRNA by weakening and destroying the last base pair of the helix HD. Weakening of the closing base pair in the stem of HD, i.e. substitution of C72G78 with AU or UA, indeed increased the binding affinity (M12 and M13). Replacing the C72G78 base pair by a non-Watson-Crick combination even restores binding of the G73C77 mutant version to wild-type levels (M10, M11). Thus, the particular backbone conformation of the three-base loop might be an important determinant for recognition of L4. One might speculate that the HD-loop does indeed only contain three nucleotides because of formation of AG or GU base pairs between positions 73 and 77 (Fig. 5) that could, in contrast to a Watson-Crick combination, enable a similar shape of the three-base loop.
Although a three-dimensional structure of the mRNA-L4 complex is clearly needed to substantiate these speculations, it is interesting to note that the backbone turn between A322 and C323, caused by the syn-conformation of the 323 base in the rRNA, is monitored in the probing results. Because of this turn, the phosphate group 5' to C323 is facing opposite to the L4 side, and is indeed the only phosphate in the loop hardly affected in the probing experiments of both the mRNA and rRNA L4 complexes (Fig. 4D, green).
Analogies between the L4 Contacts to mRNA and rRNA TargetsAlthough L4 interacts with parts of Domains I, II, IV, and V in the mature 50 S subunit (30, 38), the only part of 23 S rRNA that binds to L4 in the absence of other proteins is Domain I (15). Proposing that the two RNA targets for L4 binding have a similar structure, one would expect the same part of protein L4 to interact with each of the RNA targets.
Phylogenetically conserved amino acids in the 4/
5 regions of
L4 (Fig. 1B) contact
the RNA backbone in 23 S rRNA Domain I. In the H. marismortui
ribosome (38) (Protein Data
Bank number 1JJ1
[PDB]
), residues Lys149 (E. coli
Lys130), Thr150 (E. coli Thr131),
and Arg205 (E. coli Arg162) bridge to the three
phosphates in the A327A329 (E. coli A320A322) loop;
these contacts are compatible with phosphorothioate footprinting experiments
(Fig. 4D)
(14,
15). The only base directly
recognized in the H. marismortui rRNA-Domain I L4 binding site is
A327 (E. coli A320), which is contacted by the universally conserved
asparagine 206 (E. coli Arg163) and glutamine 151 (E.
coli Lys132). Mutations of the corresponding adenine in the HD
loop of the mRNA decrease binding affinity
(Table II, M1M3) and
affect regulation in vivo
(28).
Mutations in the 4/
5 region of E. coli L4
(Thr131, Leu134, Ala160, and
Val167) abolish L4-dependent regulation in vivo
(17). Furthermore,
modifications in the
4/
5 region of L4 from T. maritima
bestow on this protein a capacity to regulate the E. coli S10 operon
not possessed by wild-type T. maritima L4 protein
(40). Thus, amino acids in L4
that contact 23 S rRNA Domain I or are close to these L4 residues may also be
involved in recognizing the HD mRNA region, supporting the analogy between the
two binding events.
Conclusions on the Mechanism of L4-mediated Transcription
TerminationStudies of Rho-independent transcription termination
suggest that cessation or prolongation of transcription is caused by changes
in the conformation of the nascent RNA chain
(41,
42). These changes could be
effected by protein factors, such as NusA and N protein
(43,
44), or ribosomal proteins
(45). Analysis of
transcription complexes paused at other terminators
(46,
47) suggests that exactly the
minimal D-upE segment would be accessible to L4 binding in the paused complex.
L4-induced termination of transcription at the S10 attenuator requires both L4
and NusA (12,
48,
49). In the current study we
have found that L4 can bind to the S10 leader mRNA in the absence of NusA. The
effect of NusA is thus not related to L4 binding. Our footprint data suggest
changes in the RNA leader structure induced by L4 binding. If so, NusA might
be required for propagation of this signal to the RNA polymerase during the
transcription termination event.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Present address: Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Rössle-Straße 10, D-13092 Berlin-Buch, Germany. Tel.: 49-30-9406-2354; Fax: 49-30-9406-2552; E-mail: u.stelzl{at}mdc-berlin.de.
1 The abbreviations used are: r-protein, ribosomal protein; nt,
nucleotide(s); BSA, bovine serum albumin; L4EC, L4 from E. coli;
E. coli numbering is used if not indicated otherwise.
2 J. M. Zengel and L. Lindahl, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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