Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA.

Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7.

Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain.

Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site.

A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.

Analysis of the conformation of the 3' major domain of Escherichia coli16S ribosomal RNA using site-directed photoaffinity crosslinking.

The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.

Novel Gag-Pol frameshift site in human immunodeficiency virus type 1 variants resistant to protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring -1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.

Streptomycin binds to the decoding center of 16 S ribosomal RNA.

Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.

Upregulation of alpha1A- and alpha1B-adrenergic receptor mRNAs in the heart of cardiomyopathic hamsters.

An increased density of alpha1-adrenergic receptors (AR) has been linked to the development of necrotic lesions in the heart of hamsters with hereditary cardiomyopathy. To determine whether this increase results from an upregulation of the receptor mRNA(s), Northern blot analyses were carried out in the heart of 60-day-old control and cardiomyopathic hamsters using selective DNA probes for the three subtypes of alpha1-ARs (alpha1A, alpha1B and alpha1D). Transcripts for the three alpha1-ARs were detected in both control and cardiomyopathic hamsters. A two-fold increase in the alpha1A- and alpha1B-AR mRNA levels was observed in the cardiomyopathic hearts when compared to controls. In contrast, no change in the alpha1D-AR mRNA level could be detected. The enhancement in alpha1A- and alpha1B-AR mRNA levels was paralleled by a 20% increase in the total number of alpha1-ARs, as assessed by [3H]prazosin radioligand binding assays. Competition binding assays using subtype selective ligands indicated that the increased density of both alpha1A and alpha1B receptors contributes to the total alpha1-AR upregulation. Taken together, these data suggest that the early development of hereditary cardiomyopathy in hamsters is accompanied by a specific overexpression of the alpha1A- and alpha1B-ARs. A discrete increase of the alpha1-AR density could contribute to eliciting coronary microspasms, therefore participating in the development of focal necrotic lesions that are characteristic of the hamster cardiomyopathic model.

Pleiotropic effects of mutations at positions 13 and 914 in Escherichia coli 16S ribosomal RNA.

Mutations at position 13 or 914 of Escherichia coli 16S ribosomal RNA exert pleiotropic effects on protein synthesis. They interfere with the binding of streptomycin, a translational miscoding drug, to the ribosomes. They increase translational fidelity, and this effect can be related to a perturbation of the higher order structure of the 530 stem-loop, a key region for tRNA selection. In contrast, the structure of the decoding center is not perturbed. The mutations also affect translational initiation, slowing down the formation of the 30S initiation complex. This effect can be related to a destabilization of the pseudoknot helix (17-19/916-918), at the convergence of the three major domains of 16S ribosomal RNA.

Mutations at positions 13 and/or 914 in Escherichia coli 16S ribosomal RNA interfere with the initiation of protein synthesis.

Mutations at positions 13 (U-->A) and/or 914 (A-->U) of Escherichia coli 16S rRNA severely affect cell growth and protein synthesis, when expressed in vivo in a vector encoding an rrn operon under control of an inducible promoter. In vitro assays using extension inhibition indicate that the mutations interfere with the formation of the 30S translational initiation complex, which can account for their effect on cell growth. The two mutations destabilize an adjacent pseudoknot helix in which bases 17-19 pair to bases 916-918. This was shown by the increased binding of an oligodeoxyribonucleotide probe complementary to one strand of the pseudoknot helix, and by the increased reactivity to kethoxal of base G917 within this helix. These observations suggest that this pseudoknot helix participates in the formation of the 30S translational initiation complex.

Mutational and structural analysis of the RNA binding site for Escherichia coli ribosomal protein S7.

Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3' major domain of E. coli 16 S rRNA. This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G.U pair. Two hairpins with non-canonical base-pairs, 42' and 43, protrude from loops A and B, respectively. We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding. Changing the stem of hairpin 42' into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42' do not provide recognition features for S7. However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A. Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was found to be required for proper S7 binding. Moreover, replacing the U.G pair with a C.G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U.G pair is a recognition signal for S7. S7 binding was also affected by mutations in helix 29. Insertion of one nucleotide 5' to the G or 3' to the U of the G.U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G.U pair by a G.C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C.G pair upstream from the G.U pair had no effect. Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining part of loop B.

Molecular cloning and characterization of the hamster preproenkephalin A cDNA.

The cDNA for hamster preproenkephalin A (ENK) was cloned from an adrenal gland cDNA library constructed in the lambda ZapII vector. A nearly full-length cDNA was obtained and its 5' end region was completed using the technique of rapid amplification of cDNA ends (RACE). The coding and 3' untranslated regions of the hamster ENK cDNA share a high sequence identity with the rat, human, and bovine cDNAs, whereas the sequence identity is lower for the 5' untranslated region. Southern blot analysis of genomic DNA digests showed that a single copy of the ENK gene is present in the hamster haploid genome. Northern blot analysis of poly(A)+RNA from various hamster tissues indicated the following rank order for ENK messenger RNA abundance: adrenal glands > right atrium > brain > left atrium > right ventricle > ventricular septum > left ventricle, whereas primer extension analysis showed a single, identical transcriptional initiation site for the ENK mRNA in all these tissues. The sequence of the 5' untranslated region of the heart ENK cDNA was found to be identical to that from adrenal glands. This rules out the possibility that structural divergences in the 5' untranslated region of the heart ENK mRNA could decrease its translation efficiency and contribute to the very low level of enkephalin-containing peptides in the heart, compared to the adrenal glands.

Beta-adrenergic receptor desensitization in the early stage of hereditary cardiomyopathy in hamsters.

The beta-adrenergic receptor (beta AR)/adenylyl cyclase signalling pathway was examined in cardiac membranes from cardiomyopathic Syrian hamsters. Three stages were examined during the progression of this hereditary cardiomyopathy (30 days old, prenecrotic phase; 60 days old, necrotic phase; and 120 days old, compensatory phase). Isoproterenol-stimulated adenylyl cyclase activity was decreased by 32 +/- 16% in 30-day-old cardiomyopathic hamsters, compared with age-matched controls. This was not accompanied by any change in the fluoride- or forskolin-stimulated activities, suggesting that the decrease reflects a perturbation of the receptor-mediated stimulation. Neither the density nor the subcellular distribution of the beta AR, as assessed by [125I]iodocyanopindolol binding assays, was affected in these animals. However, the agonist binding properties of the beta AR were significantly affected. Indeed, the effect of guanyl nucleotides on isoproterenol binding was decreased in 30-day-old cardiomyopathic hamsters. Given that guanyl nucleotide sensitivity is correlated with the ability of the beta AR to productively interact with Gs protein, these results suggest that the decreased beta-adrenergic-stimulated adenylyl cyclase activity results from a functional uncoupling of the beta AR with no change in receptor density. The desensitization of the beta-adrenergic-stimulated adenylyl cyclase was transient, since no change in isoproterenol-stimulated adenylyl cyclase was detected in 60- and 120-day-old hamsters, compared with age-matched controls. Similarly, the receptor number and distribution were not affected at those ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Positions 13 and 914 in Escherichia coli 16S ribosomal RNA are involved in the control of translational accuracy.

Using a conditional expression system with the temperature-inducible lambda PL promoter, we previously showed that the single mutations 13U-->A and 914A-->U, and the double mutation 13U-->A and 914A-->U in Escherichia coli 16S ribosomal RNA impair the binding of streptomycin (Pinard et al., The FASEB Journal, 1993, 7, 173-176). In this study, we found that the two single mutations and the double mutation increase translational fidelity, reducing in vivo readthrough of nonsense codons and frameshifting, and decreasing in vitro misincorporation in a poly(U)-directed system. Using oligodeoxyribonucleotide probes which hybridize to the 530 loop and to the 1400 region of 16S rRNA, two regions involved in the control of tRNA binding to the A site, we observed that the mutations in rRNA increase the binding of the probe to the 530 loop but not to the 1400 region. We suggest that the mutations at positions 13 and 914 of 16S rRNA induce a conformational rearrangement in the 530 loop, which contributes to the increased accuracy of the ribosome.

Interaction of Escherichia coli ribosomal protein S7 with 16S rRNA.

The interaction between Escherichia coli ribosomal protein S7 and 16S rRNA was investigated using in vitro synthesized RNA transcripts. It was shown by nitrocellulose membrane filtration that RNA transcripts corresponding to the 3' major domain (nucleotides 926-1393) and to the lower half of this domain (nucleotides 926-986/1219-1393) bound S7 with the same affinity as 16S rRNA. A series of deletion mutants of the DNA coding for the lower half of the 3' major domain were constructed and the corresponding RNA fragments were assayed for their capacity to bind S7. A minimal domain of 108 nucleotides which can still efficiently bind S7 was thus obtained. In this domain, the 1304-1308/1329-1333 irregular helix and the 1351-1371 irregular hairpin were found to contain important determinants for S7 binding.

The 5' proximal helix of 16S rRNA is involved in the binding of streptomycin to the ribosome.

Single mutations at the end of the 5' proximal helix and in the 915 region (13U-->A or C; 914A-->U or G), and double mutations (13U-->A and 914A-->U; 13U-->C and 914A-->G) were constructed into Escherichia coli 16S ribosomal RNA. The mutations were introduced into an expression plasmid containing the rrnB operon under the transcriptional control of the temperature-inducible lambda PL promoter. None of the mutant 16S rRNAs affected cell growth when expressed. Ribosomes extracted after induction of expression of the mutant 16S rRNAs were assayed for their capacity to bind the error-inducing drug streptomycin and for translational misreading in the presence of streptomycin. All mutations impaired the binding of streptomycin, and consequently its capacity to stimulate misreading. Our results demonstrate the involvement of the 5' proximal helix of 16S rRNA in the binding of streptomycin and confirm the participation of the 915 region. They do not support a previous suggestion [Leclerc, D. and Brakier-Gingras, L. (1991) FEBS Lett., Vol. 279, pp. 171-174] that base pairing between nucleotides 13 and 914 stabilizes the binding of streptomycin.

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.

Increased preproenkephalin A gene expression in the rat heart after induction of a myocardial infarction.

The expression of preproenkephalin A (ppENK) gene was investigated in the rat heart, following the onset of myocardial infarction induced by ligation of the left anterior descending coronary artery. The relative abundance of ppENK mRNA and the level of enkephalins were measured by Northern blot analysis and radioimmunoassay, respectively, in the ventricles from control-unoperated, sham-operated, and operated rats. Three hours after the surgery, a comparison between rats with infarction and sham-operated rats revealed that the relative abundance of ppENK mRNA and the level of enkephalins were increased three- to four- and two- to three-fold, respectively, in the ventricles of rats with infarction. No difference was observed between rats with infarction and sham-operated rats 24 h after the surgery, or between rats with infarction compared at time intervals of 3 and 24 h following the surgery. The abundance of the ppENK mRNA in the polysomal fraction of the ventricular septum was also measured 3 h after the surgery and found to be threefold higher in rats with infarction as compared with sham-operated rats. These results indicate that the level of enkephalins rapidly increases in the ventricles of rats following myocardial infarction, and that this higher level may be ascribed to a stimulation of the local synthesis of enkephalins.

The interaction between streptomycin and ribosomal RNA.

The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results.

Expression of the preproenkephalin A gene in the heart of cardiomyopathic hamsters.

The expression of the preproenkephalin A gene (Enk gene), which codes for the precursor of enkephalins, was investigated in the heart of hamsters with a hereditary cardiomyopathy at four different stages of the disease: the prenecrotic stage (30 days), the necrotic stage (60 days), the hypertrophic stage (120 days), and the final stage (200 days). In control atria and ventricles, the relative abundance of the Enk mRNA, as assessed by Northern blot analysis, did not change upon ageing. In the ventricles of cardiomyopathic hamsters, however, it increased about two- to three-fold at the necrotic stage, but was unaltered at the other time points studied; whereas in the atria, it progressively decreased to reach about half that of control hamsters at the final stage. Enkephalin levels, as measured by radioimmunoassay, decreased at 60 days in both the atria and ventricles of control hamsters, and also in the atria of cardiomyopathic hamsters, and remained stable thereafter, corresponding to one-third to one-half of those at 30 days. However, in the ventricles of cardiomyopathic hamsters, the peptide level decreased only slightly, the consequence being that at 60, 120, and 200 days, it was about two- to three-fold that of control hamsters. The lack of correlation between peptide levels and the relative abundance of the Enk mRNA suggests that translational and (or) post-translational mechanisms are important in the control of the expression of the Enk gene in the heart of hamsters.

Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome.

The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed, D. and Noller, H.F. (1987) Nature, 327, 389-394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A----G and 915A----G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)- and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin.