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In this paper the question was studied whether Escherichia coli ribosomes which have undergone a specific cleavage of their 16-S rRNA by treatment with the bacteriocin cloacin DF13, are able to enter and complete an elongation cycle. As we have shown in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes with MS2 RNA as a messenger. Binding of the second aminoacyl-tRNA (alanyl-tRNA) to the latter complexes led to the formation of fMet-Ala-tRNA which was fully puromyein-sensitive. In addition to these aminoacyl-tRNAs also the third aminoacyl-tRNA (seryl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycin released all radioactivity from the ribosomes. This indicates that fMet-Ala-Ser-tRNA is formed and is translocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in a 1:1:1 ratio. The defective particles bind fMet-tRNA, Ala-tRNA and Ser-tRNA in a ratio of 1:0.5:0.25. Kinetic experiments strongly suggest that at each binding of the ternary complex aminoacyt-tRNA · EF-Tu · GTP to the A site of defective ribosomes, about half of these particles undergo irreversible inactivation. Consequently polypeptide synthesis under our conditions will come to an end after incorporation of only four or five amino acids. The particles which temporarily survive ternary complex binding are fully capable of transpeptidation and translocation. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

It is well know that protein synthesis stopped either by chloromycetin or by starvation of an essential amino acid for a RC relstrain, ribosomal RNA synthesis continues, but products do not appear in 50 S and 30 ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that: (a) during amino-acid starvation of a starvation of RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction; (b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actinomycin ;(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S parcel which contains normal 5S RNA; (d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated; (e) this precursor which is isolated from the 105000xg supernatant fraction after pulse labeling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA. [ABSTRACT FROM AUTHOR]

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with out previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A, 73, 702–706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can he compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subnnit interaction. [ABSTRACT FROM AUTHOR]

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the α-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2β) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates. [ABSTRACT FROM AUTHOR]

The mutant strain of Escherichia coli, TP28, synthesises ribosomes by an abnormal pathway and accumulates large quantities of 47S ribonucleoprotein particles. The protein complement of mutant 70S ribosomes is normal but 47S particles contain only traces of proteins L28 and L33 and have a significantly reduced content of four other proteins. The mutation reduces the rates of synthesis of L28 and L33 by about half but other widespread alterations ensue. In particular, ribosomal protein synthesis in the mutant strain becomes less well balanced than in its parent: some proteins, particularly those from promoter-proximal genes, are oversynthesized and their excess then degraded. [ABSTRACT FROM AUTHOR]

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887-895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the ’ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA. [ABSTRACT FROM AUTHOR]

Examines the molecular properties of two mutant species of the elongation factor Tu which was derived from either tuf A or tuf B. Presence of the one elongation factor in a kirromycin-resistant mutant of Escherichia coli; Competence of the elongation factors to sustain polypeptide synthesis; Interaction with guanine nucleotides; Differences that occur in the presence of antibiotic; Kirromycin resistance of the strain harboring ribosome and the elongation factor; Capability of protein synthesis.

The function of protein L1 in protein biosynthesis has been examined using ribosomes from two independently derived mutants of Escherichia coli. both of which lacked this protein. In systems in vitro with phage MS2 RNA or poly(U) as message, the mutant ribosomes showed from 40% to 60% of the activity of wild-type ribosomes. The reduction in activity was apparent in the kinetics of [14C]phenylalanine incorporation throughout the incubation period. The activities were restored fully to the wild-type level by the addition of purified protein L1. These results show on the one hand that protein L1 is not essential for protein biosynthesis but, on the other, its presence can significantly increase the overall rate of this process. The data further indicate it as likely that protein L1 exerts its effect at the elongation step. [ABSTRACT FROM AUTHOR]

It was shown that at 20 mM Mg2+ and 0 or 20 °C each 70-S ribosome binds non-enzymatically about one Phe-tRNAPhe molecule in the A site if poly(U) is used as messenger. Only a minor amount of ribosomes (never exceeding 15%) and after long incubation binds a second molecule of Phe-tRNAPhe and forms diphenylalanyl-tRNA. If the Phe-tRNAPhe preparation contains nonspecific deacylated tRNA, the latter is always bound in the P site up to one molecule per 70-S ribosome. The association constant for this non-specific tRNA binding is equal to (1.4 ± 0.3) × 107 M-1 and is independent of the messenger poly(U) and the filling of the A site by a specific aminoacyl-tRNA. It was found that the codon-dependent binding of Phe-tRNAPhe in the A site is reversible, but two different types of complexes are formed with drastic differences of binding constants. This heterogeneity is due to the presence of two conformers of Phe-tRNAPhe. The equilibrium of the two Phe-tRNAPhe conformers depends on Mg2+ and can be shifted if tRNA is heated and Mg2+ added or sequestered by complexing agents. One type of Phe-tRNAPhe yields complexes with 70-S ribosomes showing extremely high affinity constants (higher than 1011 M-1). The second conformer is characterized by a comparatively low binding constant about 5 × 106 M-1. In the binding of both species to 30-S subunits no difference is seen. A big difference in the behaviour of both Phe-tRNAPhe conformers is found also in the absence of messenger RNA. The association constant of Phe-tRNAPhe (high-affinity conformer) to 70-S ribosomes is about 3 × 108 M-1, but for the low-affinity conformer is hundred times lower. [ABSTRACT FROM AUTHOR]

Association between ribosomal subunits of Eschrichia coli can lead to the formation of two classes of 30-S · 50-S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so-called derived subunits. From cross experiments it is concluded that it is the 50-S subunit which determines the nature of the association product, whereas the 30-S subunit determines whether an association product can be formed. Native and derived 50-S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30-8 subunits lose a factor during isolation, probably IF-3. They are therefore able to associate both at 0° and 37 °C. On the other hand derived 30-S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+. A number of trivial factors have been excluded to explain the difference between the two types of 50-S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl-tRNA, known to influence subunit association. The content of tRNA and aminoacyl-tRNA has been determined quantitatively by polyacrylamide-gel electrophoresis. None of the subunits nor the 70-S ribosomes from which the derived subunits are prepared contain a tRNA species. [ABSTRACT FROM AUTHOR]

Polyribosomes containing mRNA differentially labelled with [³H]uracil and [14C]uracil in the 5′-and the 3′-proximal portions were isolated from Escherichia coli cells, mRNA degradation to alcohol-soluble products occurred when polyribosomes were incubated in conditions of protein synthesis and proceeded in the 5′ to 3′-direction. 15% of the mRNA was degraded at the end of a 10-min period of incubation. The mRNA-degrading enzyme (or enzymic system) was found to be bound to polyribosomes; it could be removed by washing in low salt. The content in RNAase activity per ribosomes in polyribosomes increased three to five-fold when polyribosomes were isolated from cells in which extensive run-off of ribosomes from mRNA had been induced. On the contrary the RNAase activity in free ribosomes was not affected. This is taken as an indication that the enzyme(s) responsible for the degradation of endogenous mRNA in vitro is not aspecifically adsorbed to ribosomes, but specifically located towards the 5′-end of the mRNA chains in polyribosomes. [ABSTRACT FROM AUTHOR]