Ribosomal System Research Articles

Mechanism of translational coupling in the nifLA operon of Klebsiella pneumoniae.

The nifLA operon of Klebsiella pneumoniae encodes the sensor-activator pair involved in the regulation of other nif genes. Balanced synthesis of both proteins, which is required for correct regulation, is achieved by coupling translation of nifA to that of nifL. The mechanism of translational coupling at the nifLA operon was analysed using a specialized ribosome system, and the effect of substituting the natural Shine-Dalgarno of nifL or nifA for specialized Shine-Dalgarno sequences was determined. Our results indicate that translational coupling occurs in this operon by a reinitiation mechanism. Additionally, reinitiation at the nifA can happen even in the absence of good Shine-Dalgarno recognition by the reinitiating ribosome, although its efficiency is lower. The effect of a putative translational enhancer sequence (downstream box) on translational coupling efficiency was also determined. Mutations that reduce the homology of the putative downstream box to the consensus had only a minor effect on nifA translation by wild-type ribosomes. However, they had a significant effect on nifA translation by specialized ribosomes, suggesting that recognition of the downstream box may compensate inefficient ribosomal interactions with the Shine-Dalgarno sequence.

Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning.

Helix 2 of the central pseudoknot structure in Escherichia coli 16S rRNA is formed by a long-distance interaction between nt 17-19 and 918-916, resulting in three base pairs: U17-A918, C18-G917and A19-U916. Previous work has shown that disruption of the central base pair abolishes ribosomal activity. We have mutated the first and last base pairs and tested the mutants for their translational activity in vivo , using a specialized ribosome system. Mutations that disrupt Watson-Crick base pairing result in strongly impaired translational activity. An exception is the mutation U916-->G, creating an A.G pair, which shows almost no decrease in activity. Mutations that maintain base complementarity have little or no impact on translational efficiency. Some of the introduced base pair substitutions substantially alter the stability of helix 2, but this does not influence ribosome functioning, neither at 42 nor at 28 degrees C. Therefore, our results do not support models in which the pseudoknot is periodically disrupted. Rather, the central pseudoknot structure is suggested to function as a permanent structural element necessary for proper organization in the center of the 30S subunit.

Antisense in abundance: the ribosome as a vehicle for antisense RNA.

Insertions at some sites within rRNA variable regions can be tolerated without affecting rRNA function. Antisense RNAs inserted at such sites in the T. thermophila rRNA can eliminate phenotypically or immunologically detectable gene expression of three genes tested. This unusually effective antisense activity is probably due to the abundance, stability and favourable intracellular localization of these antisense rRNAs with respect to mRNAs. Since antisense RNAs function very well as a part of the rRNA, rRNA might also be useful as a vehicle for other RNAs that might affect cell function such as protein binding sites or trans-acting ribozymes. The robust function of the antisense ribosome system in T. thermophila should allow the use of this system to specifically suppress gene expression and to clone genes by their null or hypomorphic phenotypes. The use of the antisense ribosome in other eukaryotes has yet to be explored, but the realization of this goal is well within the realm of possibility.

A systems process of reinforcement.

Functional systems theory was used to consider the process of reinforcement of the actions on the body of reinforcing factors, i.e., the results of behavior satisfying the body's original needs. The systems process of reinforcement includes reverse afferentation entering the CNS from receptors acted upon by various parameters of the desired results, and mechanisms for comparing reverse afferentation with the apparatus which accepts the results of the action and the corresponding emotional component. A tight interaction between reinforcement and the dominant motivation is generated on the basis of the hologram principle. Reinforcement forms an apparatus for predicting a desired result, i.e. a result-of-action acceptor. Reinforcement procedures significant changes in the activities of individual neurons in the various brain structures involved in dominant motivation, transforming their spike activity for a burst pattern to regular discharges; there are also molecular changes in neuron properties. After preliminary reinforcement, the corresponding motivation induces the ribosomal system of neurons to start synthesizing special effector molecules, which organize molecular engrams of the acceptor of the action's result. Sensory mechanisms of reinforcement are considered, with particular reference to the information role of emotions.

The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli.

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.

Amitrole Effects on Chloroplasts of Barley Plants Grown at Different Temperatures

The effects of amitrole, a bleaching herbicide affecting carotenogenesis, were studied in barley plants grown in light at 20 °C and at 30 °C. At the lower temperature the herbicide caused dramatic damage to the leaf chloroplasts, compared with control plants. The synthesis of protective carotenoids was drastically impaired, with some accumulation of lycopene and a very low production of β-carotene and xanthophylls. In chloroplasts this effect of amitrole was related to a fall in chlorophyll content, reduction of the thylakoid system and destruction of most 70S ribosomes. Though the damaged organelles maintained a certain ability to synthesize proteins, chlorophyll-binding polypeptides encoded by plastid DNA were not found in the altered thylakoids which, however, contained chlorophyll-binding proteins and other polypeptides encoded by the nuclear DNA. Amitrole appeared to be less effective in plants grown at 30 °C. The synthesis of the protective carotenoids was greatly increased and the chloroplasts exhibited structure and composition almost similar to those of control organelles. The possible existence in the carotenoid biosynthetic pathway of alternative thermo-modulated steps, with different amitrole sensitivity, is suggested.

Specialized ribosomes: highly specific translation in vivo of a single targetted mRNA species

In previous studies [Hui and de Boer, Proc. Natl. Acad. Sci. USA 84 (1987) 4762–4766; Hui et al., Methods Enzymol. 153 (1987) 432–452], it was shown that efficient translation of the human growth hormone mRNA (h GH) species having an altered Shine-Dalgarno (SD) sequence, 5′-GUGUG-3′, depends on the presence of specialized (spc) ribosomes containing the modified anti-SD (ASD) sequence, 5′-CACAC-3′, near the 3′ end of their 16S rRNA. In spite of the altered ASD sequence, spc ribosomes were not found to be committed exclusively to the translation of the h GH mRNA; no more than 30% of the total amount of protein synthesized by such ribosomes was hGH. Once we replace the coding sequence of the h GH mRNA with that of chloramphenicol acetyltransferase (CAT), the specificity of spc ribosomes for translation of a single targetted mRNA, relative to the endogeneous mRNAs, is greatly enhanced; an estimated 80% of the total amount of protein synthesized by spc ribosomes is CAT. Using the inducible spc ribosome system containing the cat gene, we show that, upon induction, spc ribosomes accumulate in large excess over the number needed for optimal translation of the targetted cat mRNA. Despite the excess, only few spc ribosomes initiate translation on a limited number of endogenous mRNAs. The excessive accumulation of spc ribosomes, which are predominantly present as free 30S subunits, is neither deleterious to the cells, nor does it lead to a feedback inhibition of the synthesis of wild-type ribosomes

Hypersensitivity of Rhodobacter sphaeroides ribosomes to protein synthesis inhibitors: structural and functional implications.

The elongation cycle of protein synthesis systems of purple nonsulfur photosynthetic bacteria Rhodobacter sphaeroides, grown both phototrophically and chemotrophically, was studied using 33 inhibitors with different chemical structures and functional and domain specificities. No functional differences between phototrophic and chemotrophic ribosomal systems were detected. Rhodobacter sphaeroides ribosomes exhibited strong hypersensitivity to nine functional inhibitors when compared with Escherichia coli ribosomes. Most of the R. sphaeroides ribosomal hypersensitivities corresponded to peptidyltransferase inhibitors, implying that this important functional neighborhood must be somehow different in the two organisms.

The mechanism of translational coupling in Escherichia coli. Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes.

Bacterial genes are commonly transcribed to form polycistronic mRNAs bearing reading frames whose respective translational efficiencies are not independently determined. As in many bacterial operons, expression of the atp genes of Escherichia coli is strongly influenced by translational coupling. The gene pair atpHA is tightly coupled, whereby atpA is translated at least three times more efficiently than atpH. However, there is no fixed stoichiometry of coupling: mutations in atpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold. We have demonstrated that secondary structure sequestering the atpA translational initiation region (TIR) is important to the coupling mechanism in that it inhibits de novo translational initiation at the atpA start codon. Genetic and structural analyses indicate that this inhibitory structure can be induced to refold into a less inhibitory conformation either by introducing two single-base substitutions or as a result of ribosomes translating atpH. We propose a model in which the secondary structure of the atpA TIR acts analogously to a "gating device" in that it restricts de novo ribosomal initiation until it is "switched" into a more open conformation. This contrasts with the function of a stem-loop structure located immediately downstream of atpA and upstream of the Shine-Dalgarno region of atpG, which was found to inhibit translation, but not to mediate tight coupling. Results obtained using the "specialized" ribosome system of Hui and de Boer ((1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4762-4766) indicate that primarily ribosomes reinitiating after termination on atpH are responsible for inducing refolding of the atpA TIR. The principle of alternative mRNA conformations with different functional properties embodied in the model presented here can only be fulfilled by certain types of structure. It is likely to operate in several steps of prokaryotic gene expression, underlying a range of regulatory events including transcriptional attenuation and translational activation.

Specialized Ribosomes Allow for the Study of Mutations in Functionally Important Regions in 16 S rRNA, without Affecting Cell Growth: The Identification of Functional Regions in the Central Domain of 16 S rRNA

In order to identify the sequences in the central domain of 16 S rRNA of Escherichia coli that are important for ribosome function, we have generated random mutations using a PCR-based mutagenesis technique. We show that the effects of such mutations on ribosomal activity can be analyzed in vivo utilizing the specialized ribosome system. With this system the effect of rRNA mutations on ribosomal activity can be studied by measuring the translation of a modified CAT-mRNA by specialized ribosomes. Specialized ribosomes do not translate the endogenous mRNAs and, therefore, are expected to constitute a nonessential pool of ribosomes within the cell. In total, we have isolated 28 different clones harboring specialized ribosomes with single or multiple point mutations. We demonstrate that for none of these clones was cell growth retarded, even though some of the mutations severely impaired the activity of the specialized ribosomes, to as low as 3% of the wild-type level. For all mutants, their individual activities ranged between 3% and 100% of that of the wild-type activity. Comparison of several mutants indicates that mutations within the hairpin loops 787-795 and 898-901 strongly reduce the ribosomal activity. We also present evidence that the single-stranded region centered around residue A815 may be involved in maintaining translational accuracy.

Spectinomycin interacts specifically with the residues G1064and C1192in 16S rRNA, thereby potentially freezing this molecule into an inactive conformation

The upper stem of helix 34, consisting of the base-paired sequences C1063G1064U1065 and A1191C1192G1193, is suggested to be involved in the binding of spectinomycin. In E. coli 16S rRNA, each of the three mutations at position C1192 confers resistance to spectinomycin. In chloroplast ribosomes from tobacco plants and algae, resistance is conferred by single mutations at positions 1064, 1191, and 1193 (E. coli numbering). Since each of these mutations disrupt any of the three basepairs in the upper stem of helix 34, it has been postulated that spectinomycin can bind to this region and inhibit protein synthesis, only if its nucleotides are basepaired. We have tested this hypothesis by introducing disruptive and compensatory mutations that alter the basepair G1064-C1192. Using the specialized ribosome system, the translational activity of such mutants was determined, in the absence and presence of spectinomycin. We show that any of the three disruptive mutations A1064, C1064, and U1064 confer resistance, in accordance with the model for spectinomycin binding. Compensatory mutations A1064U1192, C1064G1192, and U1064A1192, however, maintained the resistance. This indicates that a basepaired conformation as such is not sufficient for spectinomycin binding, but rather that a G-C pair at positions 1064 and 1192 is required. In addition, we find that the translational activity of specialized ribosomes containing the mutations C1064G1192 is 5-fold lower compared to that of ribosomes containing any of the other mutations introduced, regardless whether spectinomycin is present or not. Since the introduction of C1064G1192 is expected to increase the stability of the upper stem of helix 34, we suggest that these mutations impair ribosome function by preventing the (transient) disruption of the upper stem. By analogy, we speculate that spectinomycin blocks protein synthesis by stabilizing the upper stem. In both cases, the 30S subunit would be frozen into an inactive conformation.

Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation.

The postulated central pseudoknot formed by regions 9-13/21-25 and 17-19/916-918 of 16S rRNA of Escherichia coli is phylogenetically conserved in prokaryotic as well eukaryotic species. This pseudoknot is located at the center of the secondary structure of the 16S rRNA and connects the three major domains of this molecule. We have introduced mutations into this pseudoknot by changing the base-paired residues C18 and G917, and the effect of such mutations on the ribosomal activity was studied in vivo, using a 'specialized' ribosome system. As compared with ribosomes having the wild-type pseudoknot, the translational activity of ribosomes containing an A, G or U residue at position 18 was dramatically reduced, while the activity of mutant ribosomes having complementary bases at positions 18 and 917 was at the wild-type level. The reduced translational activity of those mutants that are incapable of forming a pseudoknot was caused by their inability to form 70S ribosomal complexes. These results demonstrate that the potential formation of a central pseudoknot in 16S rRNA with any base-paired residues at positions 18 and 917 is essential to complete the initiation process.

Functional analysis of seven ribosomal systems from extremely halophilic archaea

The sensitivity of seven extremely halophilic archaeal protein synthesis systems to 42 inhibitors with different domain (kingdom), functional, and structural specificity has been studied. The results obtained underline the functional variability of archaeal ribosomes when compared with bacterial and eukaryotic reference systems. Halophilic ribosomes are sensitive to some of the specific inhibitors of bacteria, such as thiostrepton, carbomycin A, and griseoviridin, or of eukaryotes, such as anisomycin and bruceantin, and extremely sensitive to some of the universal inhibitors, such as puromycin and sparsomycin. Suitable control experiments allow us to postulate that some negative inhibitory results may be caused by competition between positively charged antibiotics and the high ionic conditions required for halophilic protein synthesis.Key words: protein synthesis inhibitors, halophilic archaea, ribosomes.

Evaluation of a commercial DNA probe assay for the detection of Neisseria gonorrhoeae

The diagnostic value of a prototype ribosomal RNA-directed DNA probe system for Neisseria gonorrhoeae was evaluated in 119 men with urethritis. All 15 patients with positive results on Thayer-Martin culture were also positive with the DNA probe, while all 63 patients who had negative results on Thayer-Martin culture were also negative with the DNA probe. Thus the rate of agreement between the two tests was 100%. Positive DNA probe results were obtained in all 14 patients who were positive on an enzyme immunoassay, while negative DNA probe results were obtained in all 27 patients who were negative on the enzyme immunoassay. The agreement rate between these two tests was also 100%. The results of this study indicate that the DNA probe assay is a reasonable, noncultural alternative for directly detecting N gonorrhoeae in urogenital samples.

Trypanosoma cruzi 5S rRNA genes: molecular cloning, structure and chromosomal organization

To further study the ribosomal RNA genetic system in Trypanosoma cruzi, the 5S rRNA gene family was characterized. We found that this gene family is reiterated about 1600 times per diploid nuclei and is mostly organized as a tandem repeat of 481 base pairs. These gene clusters were assigned to two chromosomes of about 1500 and 1400 kilobase pairs. We found that the 5S rRNA-coding region is comprised of 120 nucleotides, and contains the well-known internal control regions of eukaryotic RNA polymerase III. The two gene-spacer regions analysed exhibit a putative signal for transcription termination and six sites homologous to the consensus sequence for the binding of transcription factor Sp1.

Testosterone effects on ribosomal RNA levels in injured peripheral motor neurons: A preliminary report

We have previously demonstrated that administration of testosterone to hamsters during the early phases of axonal regeneration following facial nerve injury accelerates both the rate of regeneration of the fastest growing population of axons and the return of functional movement. We hypothesized from those studies that testosterone primes the neuronal cell body in such a way as to accelerate the "switch" from a normal to a reparative state. That hypothesis was tested in this study using ribosomal DNA (rDNA) probes in conjunction with in situ hybridization to map the molecular response of the polymerase I system to axotomy, with and without hormone exposure. Adult male hamsters were subjected to right facial nerve severance, with the left side serving as an internal control. Half the animals were administered testosterone propionate via subcutaneous implants. In situ hybridization using a genomic rDNA probe complementary to the 28S rRNA species was accomplished, and levels of rRNA in injured facial neurons assessed both qualitatively and quantitatively. Our initial findings indicate that testosterone markedly upregulates rRNA levels after injury, and support the hypothesis of an acceleration in the metabolic switch to a reparative state. This leads us, in turn, to suggest that this effect of testosterone on the ribosomal system is causally related to the increase in axonal regeneration rate and return of functional movement previously documented in this system.

Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes

Previous studies have led to the hypothesis that some proteins of the postsynaptic membrane are locally synthesized at postsynaptic sites. To evaluate this hypothesis, synaptosome fractions that included fragments of dendrites were allowed to incorporate labeled amino acid into protein. The labeled synaptosomes were then subfractionated to the level of the synaptic plasma membrane (SPM) and then the synaptic junctional complex (SJC). The specific activity (cpm/microgram protein) of the synaptosome fraction and its subfractions was assessed by scintillation counting and protein assay, and labeled polypeptides were characterized by SDS-PAGE and fluorography. The contribution of mitochondrial and eucaryotic protein synthesis to the overall incorporation was evaluated using cycloheximide (CYC), a eucaryotic protein synthesis inhibitor, and chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor. Both the SPM and the SJC subfractions obtained from labeled synaptosomes contained labeled polypeptides. The SPM from labeled synaptosomes had a specific activity approximately equal to that of other nonmitochondrial membrane components of the synaptosome. Thus, labeling of the SPM was not due to contamination by these other labeled membrane components. The mitochondrial fraction had the highest specific activity of the membrane components of the labeled synaptosome, but the specific activity was reduced by 47% in mitochondrial fractions from CAP-treated synaptosomes, while the specific activity of the SPM was not reduced by this treatment. Thus, SPM labeling is not due to mitochondrial contamination. The specific activity of the detergent-insoluble SJC was comparable to that of the SPM from which it was derived. The possibility of labeling of SPM and SJC by contamination with soluble proteins was assessed by adding labeled soluble proteins to a cold synaptosome preparation that was then subfractionated to obtain the SPM and SJC. There was no detectable binding of labeled soluble proteins to the SPM or SJC. These results support the hypothesis that some synaptic proteins are locally synthesized. Fluorographs of SDS gels of SPM from labeled synaptosomes revealed labeled bands at approximate molecular weights of 14, 18, 26, 28, 36, 38, 42, 45, 55, 60, and 116 kDa. Six of these labeled polypeptides at 38, 42, 45, 55, 60, and 116 kDa were still evident in fluorographs of the synaptic junctional complex from labeled synaptosomes. None of these labeled bands were seen in fluorographs of SPM and SJC from CYC-treated synaptosomes, whereas they were still present in fluorographs of CAP-treated synaptosomes. These labeled polypeptides are therefore produced by eucaryotic ribosomal systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Helmichia glandulicola sp.nov. (Microspora, Thelohaniidae): morphology, development and influence on salivary glands ofChironomus anthracinus (Diptera, Chironomidae)

Helmichia glandulicola, a microsporidian parasite specific for cells of the salivary glands of the midgeChironomus anthracinus, is described. It is characterized by tubular spores with a distinct polar cap that are enclosed in persistent octosporous pansporoblasts. The pansporoblastic membrane is formed early during the first sporogonial division. A granular secretion occurs in the pansporoblast among the oval sporoblasts, which subsequently become tubular and display a rigid polar filament. The solitary infected cell reacts with hypertrophy and depletion of its endoplasmic reticulum and ribosomal system. There are differences in the morphology of mitochondria in infected vs noninfected cells. The production of saliva by the host is only partially reduced following infection.

Effect of the aqueous extract and saponin fraction from the flowers of Verbascum thapsiforme on protein biosynthesis in a rat liver ribosomal system

AbstractThe effect of fractions isolated from the aqueous extract of Verbascum flowers on protein biosynthesis was studied. A strong inhibitory effect of the aqueous extract on protein biosynthesis was demonstrated in isolated rat liver ribosomes. The saponin fraction was shown to be responsible for this activity and it was compared to commercial glycyrrhizic acid and its aglycon as the standard drungs. It was found that these compounds strongly inhibited the incorporation of [14C]leucine into proteins in vitro and that the target site for inhibition was the ribosome fraction from rat liver cells.

The Difference in the Type of Codon-Anticodon Base Pairing at the Ribosomal P-Site Is One of the Determinants of the Translational Rate1

By utilizing an enzymatically reconstructed tRNA variant containing an altered anticodon sequence, we have examined the different biochemical behavior of translation between the Watson-Crick type and the wobble type base pair interactions at the first anticodon position. We have found that the Watson-Crick type base pair has an advantage in translation in contrast to the wobble type base pair by comparing the efficiency of transpeptidation of native tRNA(Phe) (anticodon; GmAA) with its variant tRNA (anticodon; AAA) in the poly(U)-programmed ribosome system. Thomas et al. [Proc. Natl. Acad. Sci. U.S. (1988) 85, 4242-4246] showed that the wobble codon at the ribosomal A-site accepted its cognate tRNA less efficiently than the Watson-Crick base pairing codon. We report here that the wobble interaction at the ribosomal P-site also affected the rate of translation. This variable translational rate may be a mechanism of gene regulation through preferential codon usage.